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An abscisic acid (ABA) homeostasis regulated by its production, catabolism and transport in peanut leaves in response to drought stress

ABA is an important messenger that acts as the signaling mediator for regulating the adaptive response of plants to drought stress. Two production pathways, de novo biosynthesis and hydrolysis of glucose-conjugated ABA by {beta}-glucosidase (BG), increase cellular ABA levels in plants. ABA catabolism via hydroxylation by 8-hydroxylase (CYP707A), or conjugation by uridine diphosphate glucosyltransferase (UGT), decreases cellular ABA levels. The transport of ABA through ATP-binding cassette (ABC)-containing transporter proteins, members of ABC transporter G family (ABCG), across plasma membrane (PM) is another important pathway to regulate cellular ABA levels. In this study, based on our previously constructed transcriptome of peanut leaves in response to drought stress, fourteen candidate genes involved in ABA production (including AhZEP, AhNCED1 and AhNCED3, AhABA2, AhAAO1 and AhAAO2, AhABA3, AhBG11 and AhBG24), catabolism (including AhCYP707A3, AhUGT71K1 and AhUGT73B4) and transport (including AhABCG22-1 and AhABCG22-2), were identified homologously and phylogenetically, and further analyzed at the transcriptional level by real-time RT-PCR, simultaneously determining ABA levels in peanut leaves in response to drought. The high sequence identity and very similar subcellular localization of the proteins deduced from 14 identified genes involved in ABA production, catabolism and transport with the reported corresponding enzymes in databases suggest their similar roles in regulating cellular ABA levels. In response to drought stress, ABA accumulation levels in peanut leaves agree very well with the up-regulated expressions of ABA-producing genes (AhZEP, AhNCED1, AhAAO2, AhABA3, AhBG11 and AhBG24) and PM-localized ABA importer genes (AhABCG22-1 and AhABCG22-2), although the expression of ABA catabolic genes (AhCYP707A3 and AhUGT71K1) was also up-regulated. It is likely that drought-responsive induction of catabolic genes helps not only to maintain ABA levels within a permissible range, but also to prepare the plant for degradation of ABA after removal of the stress. These results suggest that ABA homeostasis in peanut leaves in response to drought may be coordinated by a master regulatory circuit that involves production, catabolism, and as well as transport.

plant biology

A high-throughput delayed fluorescence method reveals underling differences in the control of circadian rhythms in Triticum aestivum and Brassica napus

BackgroundA robust circadian clock has been implicated in plant resilience, resource-use efficiency, competitive growth and yield. A huge number of physiological processes are under circadian control in plants including: responses to biotic and abiotic stresses; flowering time; plant metabolism; and mineral uptake. Understanding how the clock functions in crops such as Triticum aestivum (bread wheat) and Brassica napus (oilseed rape) therefore has great agricultural potential. Delayed fluorescence (DF) imaging has been shown to be applicable to a wide range of plant species and requires no genetic transformation. Although DF has been used to measure period length of both mutants and wild ecotypes of Arabidopsis, this assay has never been systematically optimised for crop plants. The physical size of both B. napus and T. aestivum led us to develop a representative sampling strategy which enables high-throughput imaging of these crops.\n\nResultsIn this study, we describe the plant-specific optimisation of DF imaging to obtain reliable circadian phenotypes with the robustness and reproducibility to detect diverging periods between cultivars of the same species. We find that the age of plant material, light regime and temperature conditions all significantly effect DF rhythms and describe the optimal conditions for measuring robust rhythms in each species. We also show that sections of leaf can be used to obtain period estimates with improved throughput for larger sample size experiments.\n\nConclusionsWe present an optimized protocol for high-throughput phenotyping of circadian period specific to two economically valuable crop plants. Application of this method revealed significant differences between the periods of several widely grown elite cultivars. This method also identified intriguing differential responses of circadian rhythms in T. aestivum compared to B. napus; specifically the dramatic change to rhythm robustness when plants were imaged under constant light versus constant darkness. This points towards diverging networks underling circadian control in these two species.

plant biology

Native protein delivery into rice callus using ionic complexes of protein and cell-penetrating peptides

Direct protein delivery into intact plants remains a challenge for the agricultural and plant science fields. Cell-penetrating peptide (CPP)-mediated protein delivery requires the binding of CPPs to a protein to carry the protein into the cell through the cell wall and lipid bilayer. Thus, we prepared ionic complexes of a CPP-containing carrier peptide and a cargo protein, namely, Citrine yellow fluorescent protein, and subsequently studied their physicochemical properties. Two types of carrier peptides, BP100(KH)9 and BP100CH7, were investigated for delivery efficiency into rice callus. Both BP100(KH)9 and BP100CH7 successfully introduced Citrine protein into rice callus cells under pressure and vacuum treatment. Moreover, delivery efficiency varied at different growth stages of rice callus; 5-day rice callus was a more efficient recipient for Citrine than 21-day callus.

plant biology

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of calcium signal transduction in cyanobacteria are poorly understood, and only a few protein components are known. The current study describes a previously unreported calcium-binding protein which was called the Calcium Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind calcium, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction center-protein complexes, and downregulated respiration.

plant biology

Auxin-sensitive Aux/IAA proteins mediate drought tolerance in Arabidopsis by regulating glucosinolate levels

A detailed understanding of abiotic stress tolerance in plants is essential to provide food security in the face of increasingly harsh climatic conditions. Glucosinolates (GLSs) are secondary metabolites found in the Brassicaceae that protect plants from herbivory and pathogen attack. Here we report that in Arabidopsis, aliphatic GLS levels are regulated by the auxin-sensitive Aux/IAA repressors IAA5, IAA6, and IAA19. These proteins act in a transcriptional cascade that maintains expression of GLS levels when plants are exposed to drought conditions. Loss of IAA5/6/19 results in reduced GLS levels and decreased drought tolerance. Further, we show that this phenotype is associated with a defect in stomatal regulation. Application of GLS to the iaa5,6,19 mutants restores stomatal regulation and normal drought tolerance. GLS action is dependent on the receptor kinase GHR1, suggesting that GLS may signal via reactive oxygen species. These results provide a novel connection between auxin signaling, GLS levels and drought response.\n\nOne Sentence SummaryAux/IAA proteins promote drought tolerance by regulating glucosinolate levels.

plant biology

Convergent gene loss in aquatic plants predicts new components of plant immunity and drought response

Plant innate immunity relies on NLR receptors that recognize pathogen derived molecules and activate downstream signalling pathways. We analyzed the variation in copy number of NLR genes across flowering plants, and identified a number of species with a low number of NLRs relative to sister species. Two distinct lineages, one monocot (Lentibulariaceae) and one dicot (Alismatales) encapsulate four species with particularly few NLR genes. In these lineages, loss of NLRs coincided with loss of the well-known downstream immune signalling complex (EDS1-PAD4). When we expanded our analysis across the whole proteomes, we were able to identify other characterized immune genes absent only in Lentibulariaceae and Alismatales. Additionally, we identified a small subset of genes with unknown function convergently lost in all four species. We predicted that some of these genes may have a role in plant immunity. Gene expression analyses confirmed that a group of these genes was differentially expressed under pathogen infection. Another subset of these genes was differentially expressed upon drought providing further evidence of a link between the drought and plant immunity.

plant biology

Weed regulation by crop and grassland competition: critical biomass level and persistence rate

O_LIIt is widely agreed that competition regulates plant populations and shapes communities. Many studies have suggested that crop and grassland competition can be used for cost-effective sustainable weed control. However, effective weed management requires a precise knowledge of the effects of agronomic practices and there is a lack of quantitative indicators to compare and predict the success of weed biocontrol by competition.\nC_LIO_LIWe studied weed abundance dynamics over a 12-year period in crop-grassland rotations (rotation treatments consisted of maize, wheat and barley crops, alternating with temporary grassland maintained for three or six years in the rotation and fertilised with two different levels of nitrogen). In addition to classical statistical analysis of the different aforementioned rotation treatments, we also modelled weed abundance as a function of the crop and grassland competition, expressed here by biomasses harvested in the preceding years.\nC_LIO_LIWe show that weed abundance decreases over the years in grassland and subsequent crops only if the grassland receives sufficient nitrogen fertiliser. Our model had a much greater explanatory power than the rotation treatments. This model estimates a critical biomass level above which weeds are suppressed in subsequent years, and below which they tend to thrive. This critical biomass level was 24.3 and 4.7 tonnes ha-1 of dry matter for crops and grassland, respectively, highlighting the greater competitiveness of grasslands than of crops. Several clear differences between weed functional groups emerged.\nC_LIO_LISynthesis and applications - This new modelling approach directly links the interannual dynamics of weed populations to current and previous biomass production levels. This approach facilitates the development of environment-friendly weed management strategies and paves the way for comparisons of the competitiveness against weeds of crops and grassland under various pedoclimatic conditions and agronomic practices.\nC_LI

plant biology

A coevolved EDS1-SAG101-NRG1 module mediates cell death signaling by TIR-domain immune receptors

Plant intracellular nucleotide-binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-Interleukin1-receptor (TIR)-domain NLRs (TNLs) converge on the Enhanced Disease Susceptibility1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis TNL immunity, AtEDS1 heterodimers with Phytoalexin Deficient4 (AtPAD4) transcriptionally boost basal defense pathways. AtEDS1 uses the same surface to interact with PAD4-related Senescence-Associated Gene101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers was unclear. We show that AtEDS1-AtSAG101 function together with AtNRG1 coiled-coil domain helper NLRs as a coevolved TNL cell death signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the solanaceous species, Nicotiana benthamiana, and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants or AtPAD4-AtSAG101 chimeras identify closely aligned -helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with further components within plant species or clades to regulate downstream pathways in TNL immunity.

plant biology

Comprehensive Classification of the RNaseH-like domain-containing Proteins in Plants

BackgroundR-loop is a nucleic acid structure containing an RNA-DNA hybrid and a displaced single-stranded DNA. Recently, accumulated evidence showed that R-loops widely present in various organisms genomes and are involved in many physiological processes, including DNA replication, RNA transcription, and DNA repair. RNase H-like superfamily (RNHLS) domain-containing proteins, such as RNase H enzymes, are essential in restricting R-loop levels. However, little is known about the function and relationship of other RNHLS proteins on R-loop regulation, especially in plants. ResultsIn this study, we characterized 6193 RNHLS proteins from 13 representative plant species and clustered these proteins into 27 clusters, among which reverse transcriptases and exonucleases are the two largest groups. Moreover, we found 691 RNHLS proteins in Arabidopsis with a conserved catalytic alpha-helix and beta-sheet motif. Interestingly, each of the Arabidopsis RNHLS proteins is composed of not only an RNHLS domain but also another different protein domain. Additionally, the RNHLS genes are highly expressed in different meristems and metabolic tissues, which indicate that the RNHLS proteins might play important roles in the development and maintenance of these tissues. ConclusionsIn summary, we systematically analyzed RNHL proteins in plants and found that there are mainly 27 subclusters of them. Most of these proteins might be implicated in DNA replication, RNA transcription, and nucleic acid degradation. We classified and characterized the RNHLS proteins in plants, which may afford new insights into the investigation of novel regulatory mechanisms and functions of R-loops.

plant biology

Cytokinin functions as an asymmetric and anti-gravitropic signal in lateral roots

Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and revealed that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach revealed that two phytohormonal cues at opposite organ flanks counterbalance each others negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

plant biology

Affordable and robust phenotyping framework to analyse root system architecture of soil-grown plants

The analysis of root system growth, root phenotyping, is important to inform efforts to enhance plant resource acquisition from soils. However, root phenotyping remains challenging due to soil opacity and requires systems that optimize root visibility and image acquisition. Previously reported systems require costly and bespoke materials not available in most countries, where breeders need tools to select varieties best adapted to local soils and field conditions. Here, we present an affordable soil-based growth container (rhizobox) and imaging system to phenotype root development in greenhouses or shelters. All components of the system are made from commodity components, locally available worldwide to facilitate the adoption of this affordable technology in low-income countries. The rhizobox is large enough (~6000 cm2 visible soil) to not restrict vertical root system growth for at least seven weeks after sowing, yet light enough (~21 kg) to be routinely moved manually. Support structures and an imaging station, with five cameras covering the whole soil surface, complement the rhizoboxes. Images are acquired via the Phenotiki sensor interface, collected, stitched and analysed. Root system architecture (RSA) parameters are quantified without intervention. RSA of a dicot (chickpea, Cicer arietinum L.) and a monocot (barley, Hordeum vulgare L.) species, which exhibit contrasting root systems, were analysed. The affordable system is relevant for efforts in Ethiopia and elsewhere to enhance yields and climate resilience of chickpea and other crops for improved food security.\n\nSignificance StatementAn affordable system to characterize root system architecture of soil-grown plants was developed. Using commodity components, this will enable local efforts world-wide to breed for enhanced root systems.

plant biology

Capsidiol, a defensive sesquiterpene produced by wild tobacco in response to attack from the fungal pathogen Alternaria alternata, is regulated by an ERF2-like transcription factor

Capsidiol is a sesquiterpenoid phytoalexin produced in Nicotiana and Capsicum species in response to pathogen attack. Whether capsidiol plays a defensive role and how its biosynthesis is regulated in the wild tobacco Nicotiana attenuata when the plant is attacked by Alternaria alternata (tobacco pathotype), a notorious necrotrophic fungus causing brown spot disease, is unknown. Transcriptome analysis indicated that a metabolic switch to sesquiterpene biosynthesis occurred in young leaves of N. attenuata after A. alternata inoculation: many genes leading to sesquiterpene production were strongly up-regulated, including the capsidiol biosynthetic genes, 5-epi-aristolochene synthase (EAS) and 5-epi-aristolochene hydroxylase (EAH). Consistently, the level of capsidiol was increased dramatically in young leaves after fungal inoculation, from not detectable in mock control to 50.68 {+/-} 3.10 {micro}g/g fresh leaves at 3 days post inoculation. Capsidiol-reduced or capsidiol-depleted plants, which were generated by silencing EAHs or EASs by virus-induced gene silencing, were more susceptible to the fungus. In addition, this sesquiterpene exhibited strong anti-fungal activities against A. alternata in vitro when purified from infected plants and applied to fungal growth. Furthermore, an ERF2-like transcription factor was found to positively regulate capsidiol production and plant resistance through the direct transactivation of a capsidiol biosynthetic gene EAS12. Taken together, our results demonstrate that capsidiol, a phytoalexin highly accumulated in N. attenuata plants in response to A. alternata infection, plays an important role in pathogen resistance independent of JA and ethylene signaling pathways, and its biosynthesis is transcriptionally regulated by an ERF2-like transcription factor.\n\nHighlightOur results demonstrate that capsidiol, a phytoalexin highly accumulated in Nicotiana attenuata plants in response to Alternaria alternata attack, plays an important role in pathogen resistance, and its biosynthesis is transcriptionally regulated by an ERF2-like transcription factor.

plant biology

The role of strigolactones in regulation of stomatal conductance and plant-pathogen interactions in Arabidopsis thaliana

Strigolactones are a group of phytohormones that control shoot branching in Arabidopsis thaliana. However, in recent years they have been shown to affect many other plant processes. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria as a result of more open stomata as well as alterations in hormonal signalling. Here we show that both, strigolactone biosynthesis- (max3 and max4), and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. To search for the mechanisms that could explain pathogen- and ozone sensitivity we performed gene expression analysis and several different assays that explore the function of guard cells and regulation of guard cell signalling.\n\nTreatments with GR24 (a strigolactone analogue) resulted in very modest changes in defence-related gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4 and d14 mutants. Moreover, stomatal responses to stimuli that cause stomatal closure in wild-type plants (darkness, high CO2 and ABA) were analysed in the strigolactone mutants. In darkness both strigolactone biosynthesis and perception mutants showed reduced stomatal closure, whereas the response to high CO2 was impaired only in max2 and d14. The response to ABA was not impaired in any of the mutants. To position the role of MAX2 in the guard cell signalling network, max2 was crossed with mutants defective in ABA biosynthesis (aba2), in guard cell ABA signalling (ost1) and a scaffold protein required for proper ion channel activity (ghr1). The stomatal conductance of double mutants was consistently higher than the corresponding single mutants, suggesting that MAX2 acts in a signalling pathway that functions in parallel to the well characterized guard cell ABA signalling pathway. We propose that the impaired defence responses of max2 is related to more open stomata that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signalling (related to CO2 signalling), this protein could be one of the elusive components that allow guard cells to distinguish between different environmental conditions.

plant biology

Low temperature triggers genome-wide hypermethylation of transposable elements and centromeres in maize

Characterizing the molecular processes developed by plants to respond to environmental cues is a major task to better understand local adaptation. DNA methylation is a chromatin mark involved in the transcriptional silencing of transposable elements (TEs) and gene expression regulation. While the molecular bases of DNA methylation regulation are now well described, involvement of DNA methylation in plant response to environmental cues remains poorly characterized. Here, using the TE-rich maize genome and analyzing methylome response to prolonged cold at the chromosome and feature scales, we investigate how genomic architecture affects methylome response to stress in a cold-sensitive genotype. Interestingly, we show that cold stress induces a genome-wide methylation increase through the hypermethylation of TE sequences and centromeres. Our work highlights a cytosine context-specific response of TE methylation that depends on TE types, chromosomal location and proximity to genes. The patterns observed can be explained by the parallel transcriptional activation of multiple DNA methylation pathways that methylate TEs in the various chromatin locations where they reside. Our results open new insights into the possible role of genome-wide DNA methylation in phenotypic response to stress.

plant biology

Development of a model estimating root length density from root impacts on a soil profile in pearl millet (Pennisetum glaucum (L.) R. Br). Application to measure root system response to water stress in field conditions

Pearl millet, unlike other cereals, is able to withstand dry and hot conditions and plays an important role for food security in arid and semi-arid areas of Africa and India. However, low soil fertility and drought constrain pearl millet yield. One of the main targets to address these constraints through agricultural practices or breeding is root system architecture. In this study, in order to easily phenotype the root system in field conditions, we developed a model to predict root length density (RLD) of pearl millet plants from root intersection densities (RID) counted on a trench profile in field conditions. We identified root orientation as an important parameter to improve the relationship between RID and RLD. Root orientation was notably found to differ between thick roots (more anisotropic with depth) and fine roots (isotropic at all depths). We used our model to study pearl millet root system response to drought and showed that pearl millet reorients its root growth toward deeper soil layers that retain more water in these conditions. Overall, this model opens ways for the characterization of the impact of environmental factors and management practices on pearl millet root system development.

plant biology

Overexpression of the Rieske FeS protein of the Cytochrome b6f complex increases C4 photosynthesis

C4 plants contribute 20% to the global primary productivity despite representing only 4% of higher plant species. Their CO2 concentrating mechanism operating between mesophyll and bundle sheath cells increases CO2 partial pressure at the site of Rubisco and hence photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C4 photosynthesis. Since Cytochrome b6f is a key point of control of electron transport in C3 plants, we constitutively overexpressed the Rieske FeS subunit in Setaria viridis to study the effects on C4 photosynthesis. Rieske FeS overexpression resulted in a higher content of Cytochrome b6f in both mesophyll and bundle sheath cells without marked changes in abundances of other photosynthetic complexes and Rubisco. Plants with higher Cytochrome b6f abundance showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane. Rieske FeS abundance correlated with CO2 assimilation rate and plants with a 10% increase in Rieske FeS content showed a 10% increase in CO2 assimilation rate at ambient and saturating CO2 and high light. Our results demonstrate that Cytochrome b6f controls the rate of electron transport in C4 plants and that removing electron transport limitations can increase the rate of C4 photosynthesis.

plant biology

The gene annotated by the locus tag At3g08860 encodes a β-alanine/L-alanine aminotransferase in Arabidopsis thaliana

The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes some of which remain uncharacterized. This study elucidates the function of an uninvestigated aminotransferase annotated by the locus tag At3g08860. The cDNA was shown to functionally complement two E. coli mutants auxotrophic for the amino acids {beta}-alanine (non-proteogenic) and L-alanine (proteogenic). The elucidation of At3g08860 activity has the potential to facilitate experiments for the optimization of plant lines involved in nitrogen utilization efficiency, response to hypoxia, osmo-protection, vitamin B5 and coenzyme A metabolism.

plant biology

Timing seed germination under changing salinity: a key role of the ERECTA receptor-kinases

Appropriate timing of seed germination is crucial for the survival and propagation of plants, and for crop yield, especially in environments prone to salinity or drought. Yet, how exactly seeds perceive changes in soil conditions and integrate them to trigger germination remains elusive, especially once non-dormant. Here we report that the Arabidopsis ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERECTA-LIKE2 (ERL2) leucine-rich-repeat receptor-like kinases synergistically regulate germination and its sensitivity to salinity and osmotic stress. Loss of ER alone, or in combination with ERL1 and/or ERL2 slows down the initiation of germination and its progression to completion, or arrests it altogether until better conditions return. That function is maternally controlled via the embryo surrounding tissues, primarily the properties of the seed coat determined during seed development on the mother plant, that relate to both seed coat expansion and subsequent differentiation, particularly the formation of its mucilage. Salt-hypersensitive er, er erl1, er erl2 and triple mutant seeds also exhibit increased sensitivity to ABA during germination, and under salinity show an enhanced upregulation of the germination repressors and inducers of dormancy ABA-insensitive-3, ABA-insensitive-5, DELLA encoding RGL2 and Delay-Of-Germination-1. These findings reveal a novel role of the ERECTA kinases in the sensing of conditions at the seed surface and the integration of developmental and stress signalling pathways in seeds. They also open novel avenues for the genetic improvement of plant adaptation to harsh soils.\n\nHighlightThe ERECTA family of receptor-kinases regulates seed germination under salinity, through mucilage-mediated sensing of conditions at the seed surface, and interaction with secondary dormancy mechanisms.

plant biology