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RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.
Membrane sterol composition in Arabidopsis thaliana affects root elongation via auxin biosynthesis
(2021)
Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (beta-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.
Micro-RNAs are cellular components regulating gene expression at the post-transcription level. In the present study, artificial micro-RNAs were used to decrease the transcript level of two genes, AtExpA8 (encoding an expansin) and AHL25 (encoding an AT-hook motif nuclear localized protein) in Arabidopsis thaliana. The backbone of the Arabidopsis endogenous MIR319a micro-RNA was used in a site-directed mutagenesis approach for the generation of artificial micro-RNAs targeting two genes. The recombinant cassettes were expressed under the control of the CaMV 35S promoter in individual A. thaliana plants. Transgenic lines of the third generation were tested by isolating total RNA and by subsequent cDNA synthesis using oligo-dT18 primers and mRNAs as templates. The expression of the two target genes was checked through quantitative realtime polymerase chain reaction to confirm reduced transcript levels for AtExpA8 and AHL25. Downregulation of AtExpA8 resulted in the formation of short hypocotyls compared with those of the wild-type control in response to low pH and high salt concentration. This technology could be used to prevent the expression of exogenous and invading genes posing a threat to the normal cellular physiology of the host plant.
In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS inArabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of annbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression ofHSPgenes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation ofNBR1resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.
Coordinated cell polarization in developing tissues is a recurrent theme in multicellular organisms. In plants, a directional distribution of the plant hormone auxin is at the core of many developmental programs. A feedback regulation of auxin on the polarized localization of PIN auxin transporters in individual cells has been proposed as a self-organizing mechanism for coordinated tissue polarization, but the molecular mechanisms linking auxin signalling to PIN-dependent auxin transport remain unknown. We used a microarray-based approach to find regulators of the auxin-induced PIN relocation in Arabidopsis thaliana root, and identified a subset of a family of phosphatidylinositol transfer proteins (PITPs), the PATELLINs (PATLs). Here, we show that PATLs are expressed in partially overlapping cell types in different tissues going through mitosis or initiating differentiation programs. PATLs are plasma membrane-associated proteins accumulated in Arabidopsis embryos, primary roots, lateral root primordia and developing stomata. Higher order patl mutants display reduced PIN1 repolarization in response to auxin, shorter root apical meristem, and drastic defects in embryo and seedling development. This suggests that PATLs play a redundant and crucial role in polarity and patterning in Arabidopsis.
Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (alpha- and beta-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify beta-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels. (C) 2017 Published by Elsevier Inc.
Moderate and temporary heat stresses prime plants to tolerate, and survive, a subsequent severe heat stress. Such acquired thermotolerance can be maintained for several days under normal growth conditions, and can create a heat stress memory. We recently demonstrated that plastid-localized small heat shock protein 21 ( HSP21) is a key component of heat stress memory in Arabidopsis thaliana. A sustained high abundance of HSP21 during the heat stress recovery phase extends heat stress memory. The level of HSP21 is negatively controlled by plastid-localized metalloprotease FtsH6 during heat stress recovery. Here, we demonstrate that autophagy, a cellular recycling mechanism, exerts additional control over HSP21 degradation. Genetic and chemical disruption of both metalloprotease activity and autophagy trigger superior HSP21 accumulation, thereby improving memory. Furthermore, we provide evidence that autophagy cargo receptor ATG8-INTERACTING PROTEIN1 (ATI1) is associated with heat stress memory. ATI1 bodies co-localize with both autophagosomes and HSP21, and their abundance and transport to the vacuole increase during heat stress recovery. Together, our results provide new insights into the module for control of the regulation of heat stress memory, in which two distinct protein degradation pathways act in concert to degrade HSP21, thereby enabling cells to recover from the heat stress effect at the cost of reducing the heat stress memory.
Heat shock factor HSFA2 fine-tunes resetting of thermomemory via plastidic metalloprotease FtsH6
(2022)
The transcription factor HSFA2 fine-tunes a balance between prolongation and resetting of thermomemory in Arabidopsis via the regulation of both memory-supporting and memory-resetting genes.
Plants 'memorize' stressful events and protect themselves from future, often more severe, stresses. To maximize growth after stress, plants 'reset' or 'forget' memories of stressful situations, which requires an intricate balance between stress memory formation and the degree of forgetfulness.
HEAT SHOCK PROTEIN 21 (HSP21) encodes a small heat shock protein in plastids of Arabidopsis thaliana. HSP21 functions as a key component of thermomemory, which requires a sustained elevated level of HSP21 during recovery from heat stress. A heat-induced metalloprotease, filamentation temperature-sensitive H6 (FtsH6), degrades HSP21 to its pre-stress abundance, thereby resetting memory during the recovery phase. The transcription factor heat shock factor A2 (HSFA2) activates downstream genes essential for mounting thermomemory, acting as a positive regulator in the process.
Here, using a yeast one-hybrid screen, we identify HSFA2 as an upstream transactivator of the resetting element FtsH6. Constitutive and inducible overexpression of HSFA2 increases expression of FtsH6, whereas it is drastically reduced in the hsfa2 knockout mutant. Chromatin immunoprecipitation reveals in planta binding of HSFA2 to the FtsH6 promoter. Importantly, overexpression of HSFA2 improves thermomemory more profoundly in ftsh6 than wild-type plants.
Thus, by activating both memory-supporting and memory-resetting genes, HSFA2 acts as a cellular homeostasis factor during thermomemory.
Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.
Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways
(2017)
Mitochondria in animals are associated with development, as well as physiological and pathological behaviors. Several conserved mitochondrial genes exist between plants and higher eukaryotes. Yet, the similarities in mitochondrial function between plant and animal species is poorly understood. Here, we show that FMT (FRIENDLY MITOCHONDRIA) from Arabidopsis thaliana, a highly conserved homolog of the mammalian CLUH (CLUSTERED MITOCHONDRIA) gene family encoding mitochondrial proteins associated with developmental alterations and adult physiological and pathological behaviors, affects whole plant morphology and development under both stressed and normal growth conditions. FMT was found to regulate mitochondrial morphology and dynamics, germination, and flowering time. It also affects leaf expansion growth, salt stress responses and hyponastic behavior, including changes in speed of hyponastic movements. Strikingly, Cluh(+/-) heterozygous knockout mice also displayed altered locomotive movements, traveling for shorter distances and had slower average and maximum speeds in the open field test. These observations indicate that homologous mitochondrial genes may play similar roles and affect homologous functions in both plants and animals.
The organic electronic ion pump (OEIP) provides flow-free and accurate delivery of small signaling compounds at high spatio-temporal resolution. To date, the application of OEIPs has been limited to delivery of nonaromatic molecules to mammalian systems, particularly for neuroscience applications. However, many long-standing questions in plant biology remain unanswered due to a lack of technology that precisely delivers plant hormones, based on cyclic alkanes or aromatic structures, to regulate plant physiology. Here, we report the employment of OEIPs for the delivery of the plant hormone auxin to induce differential concentration gradients and modulate plant physiology. We fabricated OEIP devices based on a synthesized dendritic polyelectrolyte that enables electrophoretic transport of aromatic substances. Delivery of auxin to transgenic Arabidopsis thaliana seedlings in vivo was monitored in real time via dynamic fluorescent auxin-response reporters and induced physiological responses in roots. Our results provide a starting point for technologies enabling direct, rapid, and dynamic electronic interaction with the biochemical regulation systems of plants.
Leaf senescence of annual plants is a genetically programmed developmental phase. The onset of leaf senescence is however not exclusively determined by tissue age but is modulated by various environmental factors. Shading of individual attached leaves evokes dark-induced senescence. The initiation and progression of dark-induced senescence depend on the plant and the age of the affected leaf, however. In several plant species dark-induced senescence is fully reversible upon re-illumination and the leaves can regreen, but the regreening ability depends on the duration of dark incubation. We studied the ability of Arabidopsis thaliana leaves to regreen after dark-incubation with the aim to identify transcription factors (TFs) that are involved in the regulation of early dark-induced senescence and regreening. Two days shading of individual attached leaves triggers the transition into a pre-senescence state from which the leaves can largely recover. Longer periods of darkness result in irreversible senescence. Large scale qRT-PCR analysis of 1872 TF genes revealed that 649 of them are regulated in leaves during normal development, upon shading or re-illumination. Leaf shading triggered upregulation of 150 TF genes, some of which are involved in controlling senescence. Of those, 39 TF genes were upregulated after two days in the dark and regained pre-shading expression level after two days of re-illumination. Furthermore, a larger number of 422 TF genes were down regulated upon shading. In TF gene clusters with different expression patterns certain TF families are over-represented.
Plasticity in metabolism underpins local responses to nitrogen in Arabidopsis thaliana populations
(2019)
Nitrogen (N) is central for plant growth, and metabolic plasticity can provide a strategy to respond to changing N availability. We showed that two local A. thaliana populations exhibited differential plasticity in the compounds of photorespiratory and starch degradation pathways in response to three N conditions. Association of metabolite levels with growth-related and fitness traits indicated that controlled plasticity in these pathways could contribute to local adaptation and play a role in plant evolution.
The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.
Abiotic stresses cause oxidative damage in plants. Here, we demonstrate that foliar application of an extract from the seaweed Ascophyllum nodosum, SuperFifty (SF), largely prevents paraquat (PQ)-induced oxidative stress in Arabidopsis thaliana. While PQ-stressed plants develop necrotic lesions, plants pre-treated with SF (i.e., primed plants) were unaffected by PQ. Transcriptome analysis revealed induction of reactive oxygen species (ROS) marker genes, genes involved in ROS-induced programmed cell death, and autophagy-related genes after PQ treatment. These changes did not occur in PQ-stressed plants primed with SF. In contrast, upregulation of several carbohydrate metabolism genes, growth, and hormone signaling as well as antioxidant-related genes were specific to SF-primed plants. Metabolomic analyses revealed accumulation of the stress-protective metabolite maltose and the tricarboxylic acid cycle intermediates fumarate and malate in SF-primed plants. Lipidome analysis indicated that those lipids associated with oxidative stress-induced cell death and chloroplast degradation, such as triacylglycerols (TAGs), declined upon SF priming. Our study demonstrated that SF confers tolerance to PQ-induced oxidative stress in A. thaliana, an effect achieved by modulating a range of processes at the transcriptomic, metabolic, and lipid levels.
In both animal and plant kingdoms, body size is a fundamental but still poorly understood attribute of biological systems. Here we report that the Arabidopsis NAC transcription factor Regulator of Proteasomal Gene Expression' (RPX) controls leaf size by positively modulating proteasome activity. We further show that the cis-element recognized by RPX is evolutionarily conserved between higher plant species. Upon over-expression of RPX, plants exhibit reduced growth, which may be reversed by a low concentration of the pharmacological proteasome inhibitor MG132. These data suggest that the rate of protein turnover during growth is a critical parameter for determining final organ size.
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/ dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.