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Examines the hypothesis that gibberellins (GA) regulates hypocotyl growth by altering the extent of hypocotyl cell elongation in Arabidopsis thaliana. Endogenous plant growth regulators; Differences in GA dose-response relationships in light- and in dark-grown hypocotyls.

Plant survival requires an ability to adapt to differing concentrations of nutrient and toxic soil ions, yet ion sensors and associated signaling pathways are mostly unknown. Aluminum (Al) ions are highly phytotoxic, and cause severe crop yield loss and forest decline on acidic soils which represent ∼30% of land areas worldwide. Here we found an Arabidopsis mutant hypersensitive to Al. The gene encoding a leucine-rich-repeat receptor-like kinase, was named Al Resistance1 (ALR1). Al ions binding to ALR1 cytoplasmic domain recruits BAK1 co-receptor kinase and promotes ALR1-dependent phosphorylation of the NADPH oxidase RbohD, thereby enhancing reactive oxygen species (ROS) generation. ROS in turn oxidatively modify the RAE1 F-box protein to inhibit RAE1-dependent proteolysis of the central regulator STOP1, thus activating organic acid anion secretion to detoxify Al. These findings establish ALR1 as an Al ion receptor that confers resistance through an integrated Al-triggered signaling pathway, providing novel insights into ion-sensing mechanisms in living organisms, and enabling future molecular breeding of acid-soil-tolerant crops and trees, with huge potential for enhancing both global food security and forest restoration., (© 2024. The Author(s).)

Root hairs are tubular-shaped outgrowths of epidermal cells essential for plants acquiring water and nutrients from the soil. Despite their importance, the growth of root hairs is finite. How this determinate growth is precisely regulated remains largely unknown. Here we identify LONG ROOT HAIR (LRH), a GYF domain-containing protein, as a unique repressor of root hair growth. We show that LRH inhibits the association of eukaryotic translation initiation factor 4Es (eIF4Es) with the mRNA of ROOT HAIR DEFECTIVE6-LIKE4 (RSL4) that encodes the master regulator of root hair growth, repressing RSL4 translation and thus root hair elongation. RSL4 in turn directly transactivates LRH expression to maintain a proper LRH gradient in the trichoblasts. Our findings reveal a previously uncharacterized LRH-RSL4 feedback regulatory loop that limits root hair growth, shedding new light on the mechanism underlying the determinate growth of root hairs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Specific protein-protein interactions (PPIs) enable biological regulation. However, the evolution of PPI specificity is little understood. Here we trace the evolution of the land-plant growth-regulatory DELLA-SLY1/GID2 PPI, revealing progressive increase in specificity of affinity of SLY1/GID2 for a particular DELLA form. While early-diverging SLY1s display relatively broad-range DELLA affinity, later-diverging SLY1s tend towards increasingly stringent affinity for a specific DELLA A' form generated by the growth-promoting phytohormone gibberellin (GA). Our novel mutational strategy reveals amino acid substitutions contributing to the evolution of Arabidopsis thaliana SLY1 A' specificity, also showing that routes permitting reversion to broader affinity became increasingly constrained over evolutionary time. We suggest that progressive affinity narrowing may be an important evolutionary driver of PPI specificity and that increase in SLY1/GID2-DELLA specificity enabled the enhanced flexibility of plant physiological environmental adaptation conferred by the GA-DELLA growth-regulatory mechanism., (© 2023. The Author(s).)

Plant nitrogen (N)-use efficiency (NUE) is largely determined by the ability of root to take up external N sources, whose availability and distribution in turn trigger the modification of root system architecture (RSA) for N foraging. Therefore, improving N-responsive reshaping of RSA for optimal N absorption is a major target for developing crops with high NUE. In this study, we identified RNR10 (REGULATOR OF N-RESPONSIVE RSA ON CHROMOSOME 10) as the causal gene that underlies the significantly different root developmental plasticity in response to changes in N level exhibited by the indica (Xian) and japonica (Geng) subspecies of rice. RNR10 encodes an F-box protein that interacts with a negative regulator of auxin biosynthesis, DNR1 (DULL NITROGEN RESPONSE1). Interestingly, RNR10 monoubiquitinates DNR1 and inhibits its degradation, thus antagonizing auxin accumulation, which results in reduced root responsivity to N and nitrate (NO 3 - ) uptake. Therefore, modulating the RNR10-DNR1-auxin module provides a novel strategy for coordinating a desirable RSA and enhanced N acquisition for future sustainable agriculture., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell wall is the first physical barrier to aluminum (Al) toxicity. Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance; nevertheless, how it is regulated in rice remains largely unknown. In this study, we show that exogenous application of putrescines (Put) could significantly restore the Al resistance of art1, a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1 (ART1), and reduce its Al accumulation particularly in the cell wall of root tips. Based on RNA-sequencing, yeast-one-hybrid and electrophoresis mobility shift assays, we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance. Furthermore, transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30, and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation, hence reducing the Al-binding capacity of cell wall and enhancing Al resistance. Additionally, Put repressed OsMYB30 expression by eliminating Al-induced H 2 O 2 accumulation, while exogenous H 2 O 2 promoted OsMYB30 expression. We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice., (© 2022 Institute of Botany, Chinese Academy of Sciences.)

Foxtail and broomcorn millets are the most important crops in northern China since the early Neolithic. However, little evidence is available on how people managed these two crops in the past, especially in prehistory. Previous research on major C 3 crops in western Eurasia demonstrated the potential of stable carbon and nitrogen isotope analysis of charred archaeobotanical remains to reveal the management of water and manure, respectively. Here, we evaluate the feasibility of a similar approach to C 4 millets. Foxtail and broomcorn millet plants grown in pots in a greenhouse under different manuring and watering regimes were analysed to test the effects of management on stable carbon and nitrogen isotope values of grains. Stable nitrogen isotope values of both millets increased as manuring level increased, ranging from 1.7 ‰ to 5.8 ‰ in different conditions; hence, it appears a feasible tool to identify manuring practices, in agreement with results from recent field studies. However, the two millets exhibit opposing trends in stable carbon isotope values as watering level increased. The shift in stable carbon isotope values of millets is also smaller than that observed in wheat grown in the same experimental environment, making it difficult to identify millet water status archaeologically. In addition, we charred millet grains at different temperatures and for varying durations to replicate macro-botanical remains recovered archaeologically, and to evaluate the offsets in carbon and nitrogen isotope values induced by charring. We found that the stable nitrogen isotope values of foxtail millet and broomcorn millet can shift up to 1-2 ‰ when charred, while the stable carbon isotope values change less than 0.3 ‰. Overall, we demonstrate that stable nitrogen isotope values of charred foxtail and broomcorn millet seeds could provide insight into past field management practices, and both carbon and nitrogen isotope values can together inform palaeodietary reconstruction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dong, Bi, Wu, Belfield, Harberd, Christensen, Charles and Bogaard.)

Main Conclusion: Genetic analysis reveals a previously unknown role for ethylene signaling in regulating Arabidopsis thaliana nitrogen metabolism. Nitrogen (N) is essential for plant growth, and assimilation of soil nitrate (NO 3 - ) and ammonium ions is an important route of N acquisition. Although N import and assimilation are subject to multiple regulatory inputs, the extent to which ethylene signaling contributes to this regulation remains poorly understood. Here, our analysis of Arabidopsis thaliana ethylene signaling mutants advances that understanding. We show that the loss of CTR1 function ctr1-1 mutation confers resistance to the toxic effects of the NO 3 - analogue chlorate (ClO 3 - ), and reduces the activity of the nitrate reductase (NR) enzyme of NO 3 - assimilation. Our further analysis indicates that the lack of the downstream EIN2 component (conferred by novel ein2 mutations) suppresses the effect of ctr1-1, restoring ClO 3 - sensitivity and NR activity to normal. Collectively, our observations indicate an important role for ethylene signaling in regulating Arabidopsis thaliana NO 3 - metabolism. We conclude that ethylene signaling enables environmentally responsive coordination of plant growth and N metabolism., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Iron (Fe) storage in plant seeds is not only necessary for seedling establishment following germination but is also a major source of dietary Fe for humans and other animals. Accumulation of Fe in seeds is known to be low during early seed development. However, the underlying mechanism and biological significance remain elusive. Here, we show that reduced expression of Arabidopsis YABBY transcription factor INNER NO OUTER (INO) increases embryonic Fe accumulation, while transgenic overexpression of INO results in the opposite effect. INO is highly expressed during early seed development, and decreased INO expression increases the expression of NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1), which encodes a transporter that contributes to seed Fe loading. The relatively high embryonic Fe accumulation conferred by decreased INO expression is rescued by the nramp1 loss-of-function mutation. We further demonstrated that INO represses NRAMP1 expression by binding to NRAMP1-specific promoter region. Interestingly, we found that excessive Fe loading into developing seeds of ino mutants results in greater oxidative damage, leading to increased cell death and seed abortion, a phenotype that can be rescued by the nramp1 mutation. Taken together, these results indicate that INO plays an important role in safeguarding reproduction by reducing Fe loading into developing seeds by repressing NRAMP1 expression., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Phosphorus (P) is an indispensable macronutrient required for plant growth and development. Natural phosphate (Pi) reserves are finite, and a better understanding of Pi utilization by crops is therefore vital for worldwide food security. Ammonium has long been known to enhance Pi acquisition efficiency in agriculture; however, the molecular mechanisms coordinating Pi nutrition and ammonium remains unclear. Here, we reveal that ammonium is a novel initiator that stimulates the accumulation of a key regulatory protein, STOP1, in the nuclei of Arabidopsis root cells under Pi deficiency. We show that Pi deficiency promotes ammonium uptake mediated by AMT1 transporters and causes rapid acidification of the root surface. Rhizosphere acidification-triggered STOP1 accumulation activates the excretion of organic acids, which help to solubilize Pi from insoluble iron or calcium phosphates. Ammonium uptake by AMT1 transporters is downregulated by a CIPK23 protein kinase whose expression is directly modulated by STOP1 when ammonium reaches toxic levels. Taken together, we have identified a STOP1-centered regulatory network that links external ammonium with efficient Pi acquisition from insoluble phosphate sources. These findings provide a framework for developing possible strategies to improve crop production by enhancing the utilization of non-bioavailable nutrients in soil., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

The external application of nitrogen (N) fertilizers is an important practice for increasing crop production. However, the excessive use of fertilizers significantly increases production costs and causes environmental problems, making the improvement of crop N-use efficiency (NUE) crucial for sustainable agriculture in the future. Here we show that the rice (Oryza sativa) NUE quantitative trait locus DULL NITROGEN RESPONSE1 (qDNR1), which is involved in auxin homeostasis, reflects the differences in nitrate (NO3-) uptake, N assimilation, and yield enhancement between indica and japonica rice varieties. Rice plants carrying the DNR1indica allele exhibit reduced N-responsive transcription and protein abundance of DNR1. This, in turn, promotes auxin biosynthesis, thereby inducing AUXIN RESPONSE FACTOR-mediated activation of NO3- transporter and N-metabolism genes, resulting in improved NUE and grain yield. We also show that a loss-of-function mutation at the DNR1 locus is associated with increased N uptake and assimilation, resulting in improved rice yield under moderate levels of N fertilizer input. Therefore, modulating the DNR1-mediated auxin response represents a promising strategy for achieving environmentally sustainable improvements in rice yield., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Unlike most crops, in which soil acidity severely limits productivity, tea ( Camellia sinensis ) actually prefers acid soils (pH 4.0-5.5). Specifically, tea is very tolerant of acidity-promoted aluminum (Al) toxicity, a major factor that limits the yield of most other crops, and it even requires Al for optimum growth. Understanding tea Al tolerance and Al-stimulatory mechanisms could therefore be fundamental for the future development of crops adapted to acid soils. Here, we summarize the Al-tolerance mechanisms of tea plants, propose possible mechanistic explanations for the stimulation of tea growth by Al based on recent research, and put forward ideas for future crop breeding for acid soils., (© 2021 The Authors.)

Mutations are the source of both genetic diversity and mutational load. However, the effects of increasing environmental temperature on plant mutation rates and relative impact on specific mutational classes (e.g., insertion/deletion [indel] vs. single nucleotide variant [SNV]) are unknown. This topic is important because of the poorly defined effects of anthropogenic global temperature rise on biological systems. Here, we show the impact of temperature increase on Arabidopsis thaliana mutation, studying whole genome profiles of mutation accumulation (MA) lineages grown for 11 successive generations at 29°C. Whereas growth of A. thaliana at standard temperature (ST; 23°C) is associated with a mutation rate of 7 × 10 -9 base substitutions per site per generation, growth at stressful high temperature (HT; 29°C) is highly mutagenic, increasing the mutation rate to 12 × 10 -9 SNV frequency is approximately two- to threefold higher at HT than at ST, and HT-growth causes an ∼19- to 23-fold increase in indel frequency, resulting in a disproportionate increase in indels (vs. SNVs). Most HT-induced indels are 1-2 bp in size and particularly affect homopolymeric or dinucleotide A or T stretch regions of the genome. HT-induced indels occur disproportionately in nucleosome-free regions, suggesting that much HT-induced mutational damage occurs during cell-cycle phases when genomic DNA is packaged into nucleosomes. We conclude that stressful experimental temperature increases accelerate plant mutation rates and particularly accelerate the rate of indel mutation. Increasing environmental temperatures are thus likely to have significant mutagenic consequences for plants growing in the wild and may, in particular, add detrimentally to mutational load., (© 2021 Belfield et al.; Published by Cold Spring Harbor Laboratory Press.)

Because Iron (Fe) is an essential element, Fe storage in plant seeds is necessary for seedling establishment following germination. However, the mechanisms controlling seed Fe storage during seed development remain largely unknown. Here we reveal that an ERF95 transcription factor regulates Arabidopsis seed Fe accumulation. We show that expression of ERF95 increases during seed maturation, and that lack of ERF95 reduces seed Fe accumulation, consequently increasing sensitivity to Fe deficiency during seedling establishment. Conversely, overexpression of ERF95 has the opposite effects. We show that lack of ERF95 decreases abundance of FER1 messenger RNA in developing seed, which encodes Fe-sequestering ferritin. Accordingly, a fer1-1 loss-of-function mutation confers reduced seed Fe accumulation, and suppresses ERF95-promoted seed Fe accumulation. In addition, ERF95 binds to specific FER1 promoter GCC-boxes and transactivates FER1 expression. We show that ERF95 expression in maturing seed is dependent on EIN3, the master transcriptional regulator of ethylene signaling. While lack of EIN3 reduces seed Fe content, overexpression of ERF95 rescues Fe accumulation in the seed of ein3 loss-of-function mutant. Finally, we show that ethylene production increases during seed maturation. We conclude that ethylene promotes seed Fe accumulation during seed maturation via an EIN3-ERF95-FER1-dependent signaling pathway., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

Because environmentally degrading inorganic fertilizer use underlies current worldwide cereal yields, future agricultural sustainability demands enhanced nitrogen use efficiency. We found that genome-wide promotion of histone H3 lysine 27 trimethylation (H3K27me3) enables nitrogen-induced stimulation of rice tillering: APETALA2-domain transcription factor NGR5 (NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5) facilitates nitrogen-dependent recruitment of polycomb repressive complex 2 to repress branching-inhibitory genes via H3K27me3 modification. NGR5 is a target of gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1)-promoted proteasomal destruction. DELLA proteins (characterized by the presence of a conserved aspartate-glutamate-leucine-leucine-alanine motif) competitively inhibit the GID1-NGR5 interaction and explain increased tillering of green revolution varieties. Increased NGR5 activity consequently uncouples tillering from nitrogen regulation, boosting rice yield at low nitrogen fertilization levels. NGR5 thus enables enhanced nitrogen use efficiency for improved future agricultural sustainability and food security., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The indica and japonica rice (Oryza sativa) subspecies differ in nitrate (NO 3 - ) assimilation capacity and nitrogen (N) use efficiency (NUE). Here, we show that a major component of this difference is conferred by allelic variation at OsNR2, a gene encoding a NADH/NADPH-dependent NO 3 - reductase (NR). Selection-driven allelic divergence has resulted in variant indica and japonica OsNR2 alleles encoding structurally distinct OsNR2 proteins, with indica OsNR2 exhibiting greater NR activity. Indica OsNR2 also promotes NO 3 - uptake via feed-forward interaction with OsNRT1.1B, a gene encoding a NO 3 - uptake transporter. These properties enable indica OsNR2 to confer increased effective tiller number, grain yield and NUE on japonica rice, effects enhanced by interaction with an additionally introgressed indica OsNRT1.1B allele. In consequence, indica OsNR2 provides an important breeding resource for the sustainable increases in japonica rice yields necessary for future global food security.

Enhancing global food security by increasing the productivity of green revolution varieties of cereals risks increasing the collateral environmental damage produced by inorganic nitrogen fertilizers. Improvements in the efficiency of nitrogen use of crops are therefore essential; however, they require an in-depth understanding of the co-regulatory mechanisms that integrate growth, nitrogen assimilation and carbon fixation. Here we show that the balanced opposing activities and physical interactions of the rice GROWTH-REGULATING FACTOR 4 (GRF4) transcription factor and the growth inhibitor DELLA confer homeostatic co-regulation of growth and the metabolism of carbon and nitrogen. GRF4 promotes and integrates nitrogen assimilation, carbon fixation and growth, whereas DELLA inhibits these processes. As a consequence, the accumulation of DELLA that is characteristic of green revolution varieties confers not only yield-enhancing dwarfism, but also reduces the efficiency of nitrogen use. However, the nitrogen-use efficiency of green revolution varieties and grain yield are increased by tipping the GRF4-DELLA balance towards increased GRF4 abundance. Modulation of plant growth and metabolic co-regulation thus enables novel breeding strategies for future sustainable food security and a new green revolution.

Mutation is the source of genetic variation and fuels biological evolution. Many mutations first arise as DNA replication errors. These errors subsequently evade correction by cellular DNA repair, for example, by the well-known DNA mismatch repair (MMR) mechanism. Here, we determine the genome-wide effects of MMR on mutation. We first identify almost 9000 mutations accumulated over five generations in eight MMR-deficient mutation accumulation (MA) lines of the model plant species, Arabidopsis thaliana We then show that MMR deficiency greatly increases the frequency of both smaller-scale insertions and deletions (indels) and of single-nucleotide variant (SNV) mutations. Most indels involve A or T nucleotides and occur preferentially in homopolymeric (poly A or poly T) genomic stretches. In addition, we find that the likelihood of occurrence of indels in homopolymeric stretches is strongly related to stretch length, and that this relationship causes ultrahigh localized mutation rates in specific homopolymeric stretch regions. For SNVs, we show that MMR deficiency both increases their frequency and changes their molecular mutational spectrum, causing further enhancement of the GC to AT bias characteristic of organisms with normal MMR function. Our final genome-wide analyses show that MMR deficiency disproportionately increases the numbers of SNVs in genes, rather than in nongenic regions of the genome. This latter observation indicates that MMR preferentially protects genes from mutation and has important consequences for understanding the evolution of genomes during both natural selection and human tumor growth., (© 2018 Belfield et al.; Published by Cold Spring Harbor Laboratory Press.)

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii , isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions., (Copyright © 2017 by the Genetics Society of America.)

Soil salinity is one of the most commonly encountered environmental stresses affecting plant growth and crop productivity. Accordingly, plants have evolved a variety of morphological, physiological and biochemical strategies that enable them to adapt to saline growth conditions. For example, it has long been known that salinity-stress increases both the production of the gaseous stress hormone ethylene and the in planta accumulation of reactive oxygen species (ROS). Recently, there has been significant progress in understanding how the fine-tuning of ethylene biosynthesis and signaling transduction can promote salinity tolerance, and how salinity-induced ROS accumulation also acts as a signal in the mediation of salinity tolerance. Furthermore, recent advances have indicated that ethylene signaling modulates salinity responses largely via regulation of ROS-generating and ROS-scavenging mechanisms. This review focuses on these recent advances in understanding the linked roles of ethylene and ROS in salt tolerance.

Characterization of homoeallelic base-identity in allopolyploids is difficult since homeologous subgenomes are closely related and becomes further challenging if diploid-progenitor data is missing. We present HANDS2, a next-generation sequencing-based tool that enables highly accurate (>90%) genome-wide discovery of homeolog-specific base-identity in allopolyploids even in the absence of a diploid-progenitor. We applied HANDS2 to the transcriptomes of various cruciferous plants belonging to genus Brassica. Our results suggest that the three C genomes in Brassica are more similar to each other than the three A genomes, and provide important insights into the relationships between various Brassica tetraploids and their diploid-progenitors at a single-base resolution.

Coordination of shoot photosynthetic carbon fixation with root inorganic nitrogen uptake optimizes plant performance in a fluctuating environment [1]. However, the molecular basis of this long-distance shoot-root coordination is little understood. Here we show that Arabidopsis ELONGATED HYPOCOTYL5 (HY5), a bZIP transcription factor that regulates growth in response to light [2, 3], is a shoot-to-root mobile signal that mediates light promotion of root growth and nitrate uptake. Shoot-derived HY5 auto-activates root HY5 and also promotes root nitrate uptake by activating NRT2.1, a gene encoding a high-affinity nitrate transporter [4]. In the shoot, HY5 promotes carbon assimilation and translocation, whereas in the root, HY5 activation of NRT2.1 expression and nitrate uptake is potentiated by increased carbon photoassimilate (sucrose) levels. We further show that HY5 function is fluence-rate modulated and enables homeostatic maintenance of carbon-nitrogen balance in different light environments. Thus, mobile HY5 coordinates light-responsive carbon and nitrogen metabolism, and hence shoot and root growth, in a whole-organismal response to ambient light fluctuations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A subset of genes in Arabidopsis thaliana is known to be up-regulated in response to a wide range of different environmental stress factors. However, not all of these genes are characterized as yet with respect to their functions. In this study, we used transgenic knockout, overexpression and reporter gene approaches to try to elucidate the biological roles of five unknown multiple-stress responsive genes in Arabidopsis. The selected genes have the following locus identifiers: At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010. Firstly, T-DNA insertion knockout lines were identified for each locus and screened for altered phenotypes. None of the lines were found to be visually different from wildtype Col-0. Secondly, 35S-driven overexpression lines were generated for each open reading frame. Analysis of these transgenic lines showed altered phenotypes for lines overexpressing the At1g74450 ORF. Plants overexpressing the multiple-stress responsive gene At1g74450 are stunted in height and have reduced male fertility. Alexander staining of anthers from flowers at developmental stage 12-13 showed either an absence or a reduction in viable pollen compared to wildtype Col-0 and At1g74450 knockout lines. Interestingly, the effects of stress on crop productivity are most severe at developmental stages such as male gametophyte development. However, the molecular factors and regulatory networks underlying environmental stress-induced male gametophytic alterations are still largely unknown. Our results indicate that the At1g74450 gene provides a potential link between multiple environmental stresses, plant height and pollen development. In addition, ruthenium red staining analysis showed that At1g74450 may affect the composition of the inner seed coat mucilage layer. Finally, C-terminal GFP fusion proteins for At1g74450 were shown to localise to the cytosol.

Land plants have evolved adaptive regulatory mechanisms enabling the survival of environmental stresses associated with terrestrial life. Here, we focus on the evolution of the regulatory CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) component of the ethylene signaling pathway that modulates stress-related changes in plant growth and development. First, we compare CTR1-like proteins from a bryophyte, Physcomitrella patens (representative of early divergent land plants), with those of more recently diverged lycophyte and angiosperm species (including Arabidopsis [Arabidopsis thaliana]) and identify a monophyletic CTR1 family. The fully sequenced P. patens genome encodes only a single member of this family (PpCTR1L). Next, we compare the functions of PpCTR1L with that of related angiosperm proteins. We show that, like angiosperm CTR1 proteins (e.g. AtCTR1 of Arabidopsis), PpCTR1L modulates downstream ethylene signaling via direct interaction with ethylene receptors. These functions, therefore, likely predate the divergence of the bryophytes from the land-plant lineage. However, we also show that PpCTR1L unexpectedly has dual functions and additionally modulates abscisic acid (ABA) signaling. In contrast, while AtCTR1 lacks detectable ABA signaling functions, Arabidopsis has during evolution acquired another homolog that is functionally distinct from AtCTR1. In conclusion, the roles of CTR1-related proteins appear to have functionally diversified during land-plant evolution, and angiosperm CTR1-related proteins appear to have lost an ancestral ABA signaling function. Our study provides new insights into how molecular events such as gene duplication and functional differentiation may have contributed to the adaptive evolution of regulatory mechanisms in plants., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

We performed array comparative genome hybridization (aCGH) analyses of five Arabidopsis thaliana mutants with genomic deletions ranging in size from 4 bp to > 5 kb. We used the Roche NimbleGen Arabidopsis CGH 3 × 720 K whole genome custom tiling array to optimize deletion detection. Details of the microarray design and hybridization data have been deposited at the NCBI GEO repository with accession number GSE55327.

Evolution is fueled by phenotypic diversity, which is in turn due to underlying heritable genetic (and potentially epigenetic) variation. While environmental factors are well known to influence the accumulation of novel variation in microorganisms and human cancer cells, the extent to which the natural environment influences the accumulation of novel variation in plants is relatively unknown. Here we use whole-genome and whole-methylome sequencing to test if a specific environmental stress (high-salinity soil) changes the frequency and molecular profile of accumulated mutations and epimutations (changes in cytosine methylation status) in mutation accumulation (MA) lineages of Arabidopsis thaliana. We first show that stressed lineages accumulate ∼100% more mutations, and that these mutations exhibit a distinctive molecular mutational spectrum (specific increases in relative frequency of transversion and insertion/deletion [indel] mutations). We next show that stressed lineages accumulate ∼45% more differentially methylated cytosine positions (DMPs) at CG sites (CG-DMPs) than controls, and also show that while many (∼75%) of these CG-DMPs are inherited, some can be lost in subsequent generations. Finally, we show that stress-associated CG-DMPs arise more frequently in genic than in nongenic regions of the genome. We suggest that commonly encountered natural environmental stresses can accelerate the accumulation and change the profiles of novel inherited variants in plants. Our findings are significant because stress exposure is common among plants in the wild, and they suggest that environmental factors may significantly alter the rates and patterns of incidence of the inherited novel variants that fuel plant evolution., (© 2014 Jiang et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution 'nullisomic-tetrasomic' lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression., Results: We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss., Conclusions: We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.

Background: Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low., Results: Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size., Conclusions: Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.

Background: The analysis of polyploid genomes is problematic because homeologous subgenome sequences are closely related. This relatedness makes it difficult to assign individual sequences to the specific subgenome from which they are derived, and hinders the development of polyploid whole genome assemblies., Results: We here present a next-generation sequencing (NGS)-based approach for assignment of subgenome-specific base-identity at sites containing homeolog-specific polymorphisms (HSPs): 'HSP base Assignment using NGS data through Diploid Similarity' (HANDS). We show that HANDS correctly predicts subgenome-specific base-identity at >90% of assayed HSPs in the hexaploid bread wheat (Triticum aestivum) transcriptome, thus providing a substantial increase in accuracy versus previous methods for homeolog-specific base assignment., Conclusion: We conclude that HANDS enables rapid and accurate genome-wide discovery of homeolog-specific base-identity, a capability having multiple applications in polyploid genomics.

High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

Colonization of the land by multicellular green plants was a fundamental step in the evolution of life on earth. Land plants evolved from fresh-water aquatic algae, and the transition to a terrestrial environment required the acquisition of developmental plasticity appropriate to the conditions of water availability, ranging from drought to flood. Here we show that extant bryophytes exhibit submergence-induced developmental plasticity, suggesting that submergence responses evolved relatively early in the evolution of land plants. We also show that a major component of the bryophyte submergence response is controlled by the phytohormone ethylene, using a perception mechanism that has subsequently been conserved throughout the evolution of land plants. Thus a plant environmental response mechanism with major ecological and agricultural importance probably had its origins in the very earliest stages of the colonization of the land., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Sodium (Na) is ubiquitous in soils, and is transported to plant shoots via transpiration through xylem elements in the vascular tissue. However, excess Na is damaging. Accordingly, control of xylem-sap Na concentration is important for maintenance of shoot Na homeostasis, especially under Na stress conditions. Here we report that shoot Na homeostasis of Arabidopsis thaliana plants grown in saline soils is conferred by reactive oxygen species (ROS) regulation of xylem-sap Na concentrations. We show that lack of A. thaliana respiratory burst oxidase protein F (AtrbohF; an NADPH oxidase catalysing ROS production) causes hypersensitivity of shoots to soil salinity. Lack of AtrbohF-dependent salinity-induced vascular ROS accumulation leads to increased Na concentrations in root vasculature cells and in xylem sap, thus causing delivery of damaging amounts of Na to the shoot. We also show that the excess shoot Na delivery caused by lack of AtrbohF is dependent upon transpiration. We conclude that AtrbohF increases ROS levels in wild-type root vasculature in response to raised soil salinity, thereby limiting Na concentrations in xylem sap, and in turn protecting shoot cells from transpiration-dependent delivery of excess Na.

Fruit growth and development depend on highly coordinated hormonal activities. The phytohormone gibberellin (GA) promotes growth by inducing degradation of the growth-repressing DELLA proteins; however, the extent to which DELLA proteins contribute to GA-mediated gynoecium and fruit development remains to be clarified. Here, we provide an in-depth characterization of the role of DELLA proteins in Arabidopsis thaliana fruit growth. We show that DELLA proteins are key regulators of reproductive organ size and important for ensuring optimal fertilization. We demonstrate that the seedless fruit growth (parthenocarpy) observed in della mutants can be directly attributed to the constitutive activation of GA signaling. It has been known for >75 years that another hormone, auxin, can induce formation of seedless fruits. Using mutants with complete lack of DELLA activity, we show here that auxin-induced parthenocarpy occurs entirely through GA signaling in Arabidopsis. Finally, we uncover the existence of a DELLA-independent GA response that promotes fruit growth. This response requires GIBBERELLIN-INSENSITIVE DWARF1-mediated GA perception and a functional 26S proteasome and involves the basic helix-loop-helix protein SPATULA as a key component. Taken together, our results describe additional complexities in GA signaling during fruit development, which may be particularly important to optimize the conditions for successful reproduction.

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change.

The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Genetic differences between Arabidopsis thaliana accessions underlie the plant's extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions., (© 2011 Macmillan Publishers Limited. All rights reserved)

Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1-3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic ("somaclonal") variation [4-7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The phytohormone gibberellin (GA) has long been known to regulate the growth, development, and life cycle progression of flowering plants. However, the molecular GA-GID1-DELLA mechanism that enables plants to respond to GA has only recently been discovered. In addition, studies published in the last few years have highlighted previously unsuspected roles for the GA-GID1-DELLA mechanism in regulating growth response to environmental variables. Here, we review these advances within a general plant biology context and speculate on the answers to some remaining questions. We also discuss the hypothesis that the GA-GID1-DELLA mechanism enables flowering plants to maintain transient growth arrest, giving them the flexibility to survive periods of adversity.

Exposure of Arabidopsis plants to high temperature (28 degrees C) results in a dramatic change in plant development. Responses to high temperature include rapid extension of plant axes, leaf hyponasty, and early flowering. These phenotypes parallel plant responses to the threat of vegetational shade and have been shown to involve the hormone auxin. In this work, we demonstrate that high temperature-induced architectural adaptations are mediated through the bHLH transcriptional regulator PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Roles for PIF4 have previously been established in both light and gibberellin (GA) signaling, through interactions with phytochromes and DELLA proteins, respectively. Mutants deficient in PIF4 do not display elongation responses or leaf hyponasty upon transfer to high temperature. High temperature-mediated induction of the auxin-responsive gene IAA29 is also abolished in these plants. An early flowering response to high temperature is maintained in pif4 mutants, suggesting that architectural and flowering responses operate via separate signaling pathways. The role of PIF4 in temperature signaling does not, however, appear to operate through interaction with either phytochrome or DELLA proteins, suggesting the existence of a novel regulatory mechanism. We conclude that PIF4 is an important component of plant high temperature signaling and integrates multiple environmental cues during plant development.