Transcriptome analysis of iron over-accumulating Arabidopsis genotypes uncover putative novel regulators of systemic and retrograde signaling.

On account of its competence to accept and donate electrons, iron (Fe) is an essential element across all forms of life, including plants. Maintaining Fe homeostasis requires precise orchestration of its uptake, trafficking, and translocation in order to meet the demand for Fe sinks such as plastids. Plants harboring defects in the systemic Fe transporter OPT3 (OLIGOPEPTIDE TRANSPORTER 3) display constitutive Fe deficiency responses and accumulate toxic levels of Fe in their leaves. Similarly, ectopic expression of IRONMAN (IMA) genes, encoding a family of phloem-localized signaling peptides, triggers the uptake and accumulation of Fe by inhibiting the putative Fe sensor BRUTUS. This study aims at elucidating the mechanisms operating between OPT3-mediated systemic Fe transport, activation of IMA genes in the phloem, and activation of Fe uptake in the root epidermis. Transcriptional profiling of opt3-2 mutant and IMA1/IMA3 overexpressing (IMA Ox) lines uncovered a small subset of genes that were consistently differentially expressed across all three genotypes and Fe-deficient control plants, constituting potential novel regulators of cellular Fe homeostasis. In particular, expression of the the F-box protein At1g73120 was robustly induced in all genotypes, suggesting a putative function in the posttranslational regulation of cellular Fe homeostasis. As further constituents of this module, two plastid-encoded loci that putatively produce transfer ribonucleic acid (tRNA)-derived small ribonucleic acids are possibly involved in retrograde control of root Fe uptake.

L-DOPA induces iron accumulation in roots of Ipomoea aquatica and Arabidopsis thaliana in a pH-dependent manner.

BACKGROUND: Iron deficiency is the leading cause of anemia worldwide, particularly in countries with predominant plant-based diets. Plants constitute the main source of dietary iron. Increasing their iron concentration could reduce the occurrence of anemia. The water spinach Ipomoea aquatica is consumed as a vegetable throughout Asia and tolerates high iron concentrations making it an attractive candidate for iron biofortification. L-DOPA is an allelopathic molecule secreted by some legumes. L-DOPA can trigger the expression of Fe deficiency-inducible genes, and could potentially be used as a biostimulant to increase Fe concentration. RESULTS: L-DOPA significantly affected root growth of water spinach, and triggered a massive accumulation of Fe in roots. Both effects were exacerbated when L-DOPA was dissolved in KOH, which is surprising given that L-DOPA is less stable at high pH. To check whether a higher pH could indeed increase the bioactivity of L-DOPA, we used Arabidopsis thaliana, which grows at lower pH than water spinach, and subjected the plants to L-DOPA treatments at pH 5.5 and pH 6.0, which are both within the optimal range for Arabidopsis nutrition. At pH 6.0, the root growth of Arabidopsis was more strongly inhibited than at pH 5.5. We found that at higher pH, L-DOPA oxidizes to form a melanin precipitate. CONCLUSIONS: We concluded that the oxidation of L-DOPA that we observed upon solubilization in KOH, or in nutrient solutions at slightly higher pH produces melanin-related molecules that are more potent than L-DOPA itself to trigger the primary root growth inhibition, Fe uptake and root Fe accumulation in water spinach and Arabidopsis.

Alkalinity modulates a unique suite of genes to recalibrate growth and pH homeostasis.

Alkaline soils pose a conglomerate of constraints to plants, restricting the growth and fitness of non-adapted species in habitats with low active proton concentrations. To thrive under such conditions, plants have to compensate for a potential increase in cytosolic pH and restricted softening of the cell wall to invigorate cell elongation in a proton-depleted environment. To discern mechanisms that aid in the adaptation to external pH, we grew plants on media with pH values ranging from 5.5 to 8.5. Growth was severely restricted above pH 6.5 and associated with decreasing chlorophyll levels at alkaline pH. Bicarbonate treatment worsened plant performance, suggesting effects that differ from those exerted by pH as such. Transcriptional profiling of roots subjected to short-term transfer from optimal (pH 5.5) to alkaline (pH 7.5) media unveiled a large set of differentially expressed genes that were partially congruent with genes affected by low pH, bicarbonate, and nitrate, but showed only a very small overlap with genes responsive to the availability of iron. Further analysis of selected genes disclosed pronounced responsiveness of their expression over a wide range of external pH values. Alkalinity altered the expression of various proton/anion co-transporters, possibly to recalibrate cellular proton homeostasis. Co-expression analysis of pH-responsive genes identified a module of genes encoding proteins with putative functions in the regulation of root growth, which appears to be conserved in plants subjected to low pH or bicarbonate. Our analysis provides an inventory of pH-sensitive genes and allows comprehensive insights into processes that are orchestrated by external pH.

Arabidopsis Micro-grafting to Study the Systemic Signaling of Nutrient Status.

Grafting enables the study of systemic signals that plants use to maintain their homeostasis at the level of the whole organism. Several protocols of Arabidopsis grafting have been published over the years. These methods are limited because they either affect the overall behavior of the plant, or their throughput is low. The method presented here is based on grafting 3- to 4-days-old seedlings directly on an agar plate, without the use of hormone or collar, and can produce consistently over a hundred grafted plants per day and operator.

Genomically Hardwired Regulation of Gene Activity Orchestrates Cellular Iron Homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient which plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homoeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.

New aspects of iron-copper crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana.

Iron (Fe) and copper (Cu) are essential micronutrients for energy metabolism and reactive oxygen species (ROS) scavenging. Some Cu-containing proteins can be substituted with Fe-containing proteins, and vice versa, while several Arabidopsis genes are regulated by both metals. Few details of how plants coordinate Fe-Cu crosstalk are known. Gene expression was measured in the roots and rosettes of Fe, Cu, and simultaneously Fe and Cu deficient WT plants and a mutant of the Cu-uptake transcription factor SPL7. The spl7 mutant accumulated excess Fe under normal conditions, and lower Fe supply rescued the growth phenotype and normalized the Fe : Cu ratios. Most Fe regulated genes were expressed similarly in the WT and spl7 mutant, although at higher fold-change levels in spl7 mutants. Expression patterns indicated that both SPL7 and the FIT Fe uptake transcription factor influenced the expression of many key Fe uptake genes. Most notably, the newly discovered IMA/FEP genes and the subgroup Ib bHLH genes, which are upstream of Fe uptake responses, were repressed in the WT under Cu deficiency. Several AP2/ethylene response factor (AP2/ERF) genes and other redox homeostasis network genes were derepressed in spl7 mutants. Together, we present new information about Fe-Cu crosstalk in plants that could be applied for developing abiotic stress tolerant crops.

Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis.

Iron (Fe) is an essential mineral that has low solubility in alkaline soils, where its deficiency results in chlorosis. Whether low Fe supply and alkaline pH stress are equivalent is unclear, as they have not been treated as separate variables in molecular physiological studies. Additionally, molecular responses to these stresses have not been studied in leaf and root tissues simultaneously. We tested how plants with the Strategy I Fe uptake system respond to Fe deficiency at mildly acidic and alkaline pH by measuring root ferric chelate reductase (FCR) activity and expression of selected Fe uptake genes and riboflavin synthesis genes. Alkaline pH increased cucumber (Cucumis sativus L.) root FCR activity at full Fe supply, but alkaline stress abolished FCR response to low Fe supply. Alkaline pH or low Fe supply resulted in increased expression of Fe uptake genes, but riboflavin synthesis genes responded to Fe deficiency but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but alkaline stress blocked up-regulation of these genes in Fe-deficient leaves. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These results suggest a model implicating shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress.

Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana.

AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal aperture of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B, PLC, P5CS1, cpHSC70 s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis.

Drought and salt stress tolerance of an Arabidopsis glutathione S-transferase U17 knockout mutant are attributed to the combined effect of glutathione and abscisic acid.

Although glutathione S-transferases (GSTs) are thought to play major roles in oxidative stress metabolism, little is known about the regulatory functions of GSTs. We have reported that Arabidopsis (Arabidopsis thaliana) GLUTATHIONE S-TRANSFERASE U17 (AtGSTU17; At1g10370) participates in light signaling and might modulate various aspects of development by affecting glutathione (GSH) pools via a coordinated regulation with phytochrome A. Here, we provide further evidence to support a negative role of AtGSTU17 in drought and salt stress tolerance. When AtGSTU17 was mutated, plants were more tolerant to drought and salt stresses compared with wild-type plants. In addition, atgstu17 accumulated higher levels of GSH and abscisic acid (ABA) and exhibited hyposensitivity to ABA during seed germination, smaller stomatal apertures, a lower transpiration rate, better development of primary and lateral root systems, and longer vegetative growth. To explore how atgstu17 accumulated higher ABA content, we grew wild-type plants in the solution containing GSH and found that they accumulated ABA to a higher extent than plants grown in the absence of GSH, and they also exhibited the atgstu17 phenotypes. Wild-type plants treated with GSH also demonstrated more tolerance to drought and salt stresses. Furthermore, the effect of GSH on root patterning and drought tolerance was confirmed by growing the atgstu17 in solution containing l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH biosynthesis. In conclusion, the atgstu17 phenotype can be explained by the combined effect of GSH and ABA. We propose a role of AtGSTU17 in adaptive responses to drought and salt stresses by functioning as a negative component of stress-mediated signal transduction pathways.

Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response.

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.