The effects of copper deficiency on lignification, xylem vessel structure, and hydraulic traits in hybrid poplar.

Copper (Cu) homeostasis is integral to many plant physiological processes, including lignification of plant cell walls. This link occurs through Cu's role as a cofactor in the apoplastic laccase enzymes that oxidize monolignols that then polymerize to form the hydrophobic lignin polymer, which provides rigidity and strength to the water transport system. In this study, we investigated the effect of Cu deficiency on lignin content and chemistry in poplar stems. We also examined the effect of Cu deficiency on the stiffness of stem wood and the hydraulic properties of leaves. Cu deficiency resulted in a significant reduction in lignin content, an increase in the syringyl to guaiacyl monomer ratio of stem xylem, and no change to stem modulus of elasticity. Accompanying these stem traits, Cu-deficient leaves had higher (less negative) turgor loss points and markedly stiffer mesophyll cell walls. Our results may reflect a novel response in poplar whereby structural stiffness and mechanical stability are maintained in the face of Cu deficiency and reduction in the guaiacyl lignin monomer content.

Age Dependent Partitioning Patterns of Essential Nutrients Induced by Copper Feeding Status in Leaves and Stems of Poplar.

Copper (Cu) is an essential micronutrient, and its deficiency can cause plants to undergo metabolic changes at several levels of organization. It has been shown that leaf age can play a role in nutrient partitioning along the shoot axis of poplar. In this study, we investigated the effect of Cu deficiency on the altered partitioning of essential macro and micronutrients in leaves and stems of different age. Cu deficiency was associated with higher concentrations of calcium, magnesium, sulfur, iron, zinc, manganese, and molybdenum in leaves and relatively higher concentrations of calcium, phosphorus, iron, and zinc in stems. Leaf and stem age had significant effects on nutrient partitioning. Principal component analyses revealed patterns that point to inverse influences in leaves and stems on nutrient partitioning. Specifically, these analyses revealed that nutrient partitioning in leaves was influenced by Cu feeding status more than developmental stage, whereas nutrient partitioning in stems was influenced by developmental stage more than Cu feeding status. These results suggest that Cu deficiency and developmental stage can significantly influence the partitioning and homeostasis of macro and micronutrients in poplar organs.

Recovery after deficiency: systemic copper prioritization and partitioning in the leaves and stems of hybrid poplar.

Copper (Cu) is important for many aspects of plant function including photosynthesis. It has been suggested that photosynthesis, especially in young leaves, is prioritized for Cu delivery after deficiency in hybrid poplar. To determine relative Cu delivery prioritization, we enriched hydroponic plant growth media of Cu-deficient poplar with 98% 65Cu and tracked Cu delivery after deficiency to young leaves, mature leaves and stems. Young leaves acquired ~58% more 65Cu on Day 1 and ~65% more 65Cu by Day 3 compared with mature leaves. Additionally, stomatal conductance (gs) was measured on leaves for 6 weeks and during a 3-day 65Cu pulse resupply period. During deficiency, mature leaves maintained a higher gs than younger leaves but 3 days after Cu resupply the younger leaves that had recovered showed the highest gs. In conclusion, these results provide a quantitative understanding of how Cu is systemically transported and distributed to photosynthetic and stem tissues.

Iron deficiency and the loss of chloroplast iron-sulfur cluster assembly trigger distinct transcriptome changes in Arabidopsis rosettes.

Regulation of mRNA abundance revealed a genetic program for plant leaf acclimation to iron (Fe) limitation. The transcript for SUFB, a key component of the plastid iron-sulfur (Fe-S) assembly pathway is down-regulated early after Fe deficiency, and prior to down-regulation of mRNAs encoding abundant chloroplast Fe containing proteins, which should economize the use of Fe. What controls this system is unclear. We utilized RNA-seq. aimed to identify differentially expressed transcripts that are co-regulated with SUFB after Fe deficiency in leaves. To distinguish if lack of Fe or lack of Fe-S cofactors and associated loss of enzymatic and photosynthetic activity trigger transcriptome reprogramming, WT plants on low Fe were compared with an inducible sufb-RNAi knockdown. Fe deficiency targeted a limited set of genes and predominantly affected transcripts for chloroplast localized proteins. A set of glutaredoxin transcripts was concertedly down-regulated early after Fe deficiency, however when these same genes were down-regulated by RNAi the effect on known chloroplast Fe deficiency marker proteins was minimal. In promoters of differentially expressed genes, binding motifs for AP2/ERF transcription factors were most abundant and three AP2/ERF transcription factors were also differentially expressed early after low Fe treatment. Surprisingly, Fe deficiency in a WT on low Fe and a sufb-RNAi knockdown presented very little overlap in differentially expressed genes. sufb-RNAi produced expression patterns expected for Fe excess and up-regulation of a transcript for another Fe-S assembly component not affected by low Fe. These findings indicate that Fe scarcity, not Fe utilization, triggers reprogramming of the transcriptome in leaves.

Micronutrient homeostasis and chloroplast iron protein expression is largely maintained in a chloroplast copper transporter mutant.

PAAI is a P-Type ATPase that functions to import copper (Cu) into the chloroplast. Arabidopsis thaliana (L.) Heynh. paa1 mutants have lowered plastocyanin levels, resulting in a decreased photosynthetic electron transport rate. In nature, iron (Fe) and Cu homeostasis are often linked and it can be envisioned that paa1 acclimates its photosynthetic machinery by adjusting expression of its chloroplast Fe-proteome, but outside of Cu homeostasis paa1 has not been studied. Here, we characterise paa1 ultrastructure and accumulation of electron transport chain proteins in a paa1 allelic series. Furthermore, using hydroponic growth conditions, we characterised metal homeostasis in paa1 with an emphasis on the effects of Fe deficiency. Surprisingly, the paa1 mutation does not affect chloroplast ultrastructure or the accumulation of other photosynthetic electron transport chain proteins, despite the strong decrease in electron transport rate. The regulation of Fe-related photosynthetic electron transport proteins in response to Fe status was maintained in paa1, suggesting that regulation of the chloroplast Fe proteins ignores operational signals from photosynthetic output. The characterisation of paa1 has revealed new insight into the regulation of expression of the photosynthetic electron transport chain proteins and chloroplast metal homeostasis and can help to develop new strategies for the detection of shoot Fe deficiency.

Regulation of Iron Homeostasis and Use in Chloroplasts.

Iron (Fe) is essential for life because of its role in protein cofactors. Photosynthesis, in particular photosynthetic electron transport, has a very high demand for Fe cofactors. Fe is commonly limiting in the environment, and therefore photosynthetic organisms must acclimate to Fe availability and avoid stress associated with Fe deficiency. In plants, adjustment of metabolism, of Fe utilization, and gene expression, is especially important in the chloroplasts during Fe limitation. In this review, we discuss Fe use, Fe transport, and mechanisms of acclimation to Fe limitation in photosynthetic lineages with a focus on the photosynthetic electron transport chain. We compare Fe homeostasis in Cyanobacteria, the evolutionary ancestors of chloroplasts, with Fe homeostasis in green algae and in land plants in order to provide a deeper understanding of how chloroplasts and photosynthesis may cope with Fe limitation.

Conserved Cu-MicroRNAs in Arabidopsis thaliana Function in Copper Economy under Deficiency.

Copper (Cu) is a micronutrient for plants. Three small RNAs, which are up-regulated by Cu deficiency and target transcripts for Cu proteins, are among the most conserved microRNAs in plants. It was hypothesized that these Cu-microRNAs help save Cu for the most essential Cu-proteins under deficiency. Testing this hypothesis has been a challenge due to the redundancy of the Cu microRNAs and the properties of the regulatory circuits that control Cu homeostasis. In order to investigate the role of Cu-microRNAs in Cu homeostasis during vegetative growth, we used a tandem target mimicry strategy to simultaneously inhibit the function of three conserved Cu-microRNAs in Arabidopsis thaliana. When compared to wild-type, transgenic lines that express the tandem target mimicry construct showed reduced Cu-microRNA accumulation and increased accumulation of transcripts that encode Cu proteins. As a result, these mimicry lines showed impaired photosynthesis and growth compared to wild type on low Cu, which could be ascribed to a defect in accumulation of plastocyanin, a Cu-containing photosynthetic electron carrier, which is itself not a Cu-microRNA target. These data provide experimental support for a Cu economy model where the Cu-microRNAs together function to allow maturation of essential Cu proteins under impending deficiency.

Expression of the Intracellular COPT3-Mediated Cu Transport Is Temporally Regulated by the TCP16 Transcription Factor.

Copper is an essential element in plants. When scarce, copper is acquired from extracellular environment or remobilized from intracellular sites, through members of the high affinity copper transporters family COPT located at the plasma membrane and internal membrane, respectively. Here, we show that COPT3 is an intracellular copper transporter, located at a compartment of the secretory pathway, that is mainly expressed in pollen grains and vascular bundles. Contrary to the COPT1 plasma membrane member, the expression of the internal COPT3 membrane transporter was higher at 12 h than at 0 h of a neutral photoperiod day under copper deficiency. The screening of a library of conditionally overexpressed transcription factors implicated members of the TCP family in the COPT3 differential temporal expression pattern. Particularly, in vitro, TCP16 was found to bind to the COPT3 promoter and down-regulated its expression. Accordingly, TCP16 was mainly expressed at 0 h under copper deficiency and induced at 12 h by copper excess. Moreover, TCP16 overexpression resulted in increased sensitivity to copper deficiency, whereas the tcp16 mutant was sensitive to copper excess. Both copper content and the expression of particular copper status markers were altered in plants with modified levels of TCP16. Consistent with TCP16 affecting pollen development, the lack of COPT3 function led to altered pollen morphology. Furthermore, analysis of copt3 and COPT3 overexpressing plants revealed that COPT3 function exerted a negative effect on TCP16 expression. Taken together, these results suggest a differential daily regulation of copper uptake depending on the external and internal copper pools, in which TCP16 inhibits copper remobilization at dawn through repression of intracellular transporters.

Comparison of ATP sulfurylase 2 from selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata reveals differential intracellular localization and enzyme activity levels.

BACKGROUND: The plant Stanleya pinnata hyperaccumulates Se up to 0.5% of its dry weight in organic forms, whereas the closely related Stanleya elata does not hyperaccumulate Se. ATP sulfurylase (ATPS) can catalyze the formation of adenosine 5'-phosphoselenate (APSe) from ATP and selenate. We investigated the S. pinnata ATPS2 isoform (SpATPS2) to assess its possible role in Se hyperaccumulation. METHODS: ATPS expression and activity was compared in the two Stanleya species. The ATPS2 protein sequences were modeled. Sub-cellular locations were analyzed using GFP fusions. Enzyme activity of purified recombinant SpATPS2 was measured. RESULTS: ATPS2 transcript levels were six-fold higher in roots of S. pinnata relative to S. elata. Overall root ATPS enzyme activity was two-fold elevated in S. pinnata. Cloning and sequencing of SpATPS2 and S. elata ATPS2 (SeATPS2) showed the predicted SeATPS2 to be canonical, while SpATPS2, although very similar in its core structure, has unique features, including an interrupted plastid targeting signal due to a stop codon in the 5' region of the coding sequence. Indeed GFP fusions revealed that SpATPS2 had exclusive cytosolic localization, while SeATPS2 showed dual localization in plastids and cytosol. SpATPS2 activity was inhibited by both sulfate and selenate, indicating that the enzyme acts on both substrates. CONCLUSIONS: The ATPS2 from S. pinnata differs from non-accumulator ATPS2 in its elevated expression and sub-cellular localization. It likely acts on both selente and sulfate substrates. GENERAL SIGNIFICANCE: These observations shed new light on the role of ATPS2 in the evolution of Se hyperaccumulation in plants. This article is part of a Special Issue entitled Selenium research in biochemistry and biophysics - 200 year anniversary issue, edited by Dr. Elias Arnér and Dr. Regina Brigelius-Flohe.

Influence of sulfate supply on selenium uptake dynamics and expression of sulfate/selenate transporters in selenium hyperaccumulator and nonhyperaccumulator Brassicaceae.

Stanleya pinnata not only hyperaccumulates selenium (Se) to 0.5% of its dry weight, but also exhibits higher tissue Se-to-sulfur (S) ratios than other species and its surroundings. To investigate the mechanisms underlying this Se enrichment, we compared S. pinnata with the nonhyperaccumulators S. elata and Brassica juncea for selenate uptake in long- (9 d) and short-term (1 h) assays, using different concentrations of selenate and competitor sulfate. Different sulfate pre-treatments (0, 0.5, 5 mM, 3 d) were also tested for effects on selenate uptake and sulfate transporters' expression. Relative to nonhyperaccumulators, S. pinnata showed higher rates of root and shoot Se accumulation and less competitive inhibition by sulfate or by high-S pretreatment. The selenate uptake rate for S. pinnata (1 h) was three- to four-fold higher than for nonhyperaccumulators, and not significantly affected by 100-fold excess sulfate, which reduced selenate uptake by 100% in S. elata and 40% in B. juncea. Real-time reverse transcription PCR indicated constitutive upregulation in S. pinnata of sulfate transporters SULTR1;2 (root influx) and SULTR2;1 (translocation), but reduced SULTR1;1 expression (root influx). In S. pinnata, selenate uptake and translocation rates are constitutively elevated and relatively sulfate-independent. Underlying mechanisms likely include overexpression of SULTR1;2 and SULTR2;1, which may additionally have evolved enhanced specificity for selenate over sulfate.

A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis (Arabidopsis thaliana) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b6f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency.

The copper microRNAs.

1030 I. 1030 II. 1030 III. 1031 IV. 1031 V. 1032 VI. 1033 VII. 1034 VIII. 1034 1034 References 1034 SUMMARY: Copper (Cu) microRNAs are upregulated by Cu deficiency and mediate the post-transcriptional downregulation of transcripts that encode Cu proteins, suggesting a role directly related to Cu. However, expression and phenotypic analyses of copper microRNA mutants and over-expressors have suggested roles mainly in tolerance to abiotic stresses. To reconcile available data, a model is proposed which emphasizes the mobile nature of copper microRNA molecules in the regulation of Cu homeostasis. It is proposed that the Cu-microRNA regulatory circuits are further co-opted by plants to regulate both beneficial and pathogenic interactions with microbes. Further exploration of Cu-microRNA functions that account for the cell-to-cell mobility should give novel insight into plant microbe interactions and the integration of micronutrition and development.

Superoxide generated from the glutathione-mediated reduction of selenite damages the iron-sulfur cluster of chloroplastic ferredoxin.

Selenium assimilation in plants is facilitated by several enzymes that participate in the transport and assimilation of sulfate. Manipulation of genes that function in sulfur metabolism dramatically affects selenium toxicity and accumulation. However, it has been proposed that selenite is not reduced by sulfite reductase. Instead, selenite can be non-enzymatically reduced by glutathione, generating selenodiglutathione and superoxide. The damaging effects of superoxide on iron-sulfur clusters in cytosolic and mitochondrial proteins are well known. However, it is unknown if superoxide damages chloroplastic iron-sulfur proteins. The goals of this study were twofold: to determine whether decreased activity of sulfite reductase impacts selenium tolerance in Arabidopsis, and to determine if superoxide generated from the glutathione-mediated reduction of selenite damages the iron-sulfur cluster of ferredoxin. Our data demonstrate that knockdown of sulfite reductase in Arabidopsis does not affect selenite tolerance or selenium accumulation. Additionally, we provide in vitro evidence that the non-enzymatic reduction of selenite damages the iron-sulfur cluster of ferredoxin, a plastidial protein that is an essential component of the photosynthetic light reactions. Damage to ferredoxin's iron-sulfur cluster was associated with formation of apo-ferredoxin and impaired activity. We conclude that if superoxide damages iron-sulfur clusters of ferredoxin in planta, then it might contribute to photosynthetic impairment often associated with abiotic stress, including toxic levels of selenium.

Regulation of Cu delivery to chloroplast proteins.

Plastocyanin is a copper (Cu)-requiring protein that functions in photosynthetic electron transport in the thylakoid lumen of plants. To allow plastocyanin maturation, Cu must first be transported into the chloroplast stroma by means of the PAA1/HMA6 transporter and then into the thylakoid lumen by the PAA2/HMA8 transporter. Recent evidence indicated that the chloroplast regulates Cu transport into the thylakoids via Clp protease-mediated turnover of PAA2/HMA8. Here we present further genetic evidence that this regulatory mechanism for the adjustment of intra-cellular Cu distribution depends on stromal Cu levels. A key transcription factor mediating Cu homeostasis in plants is SQUAMOSA promoter binding protein-like7 (SPL7). SPL7 transcriptionally regulates Cu homeostasis when the nutrient becomes limiting by up-regulating expression of Cu importers at the cell membrane, and down-regulating expression of seemingly non-essential cuproproteins. It was proposed that this latter mechanism favors Cu delivery to the chloroplast. We propose a 2-tiered system which functions to control plant leaf Cu homeostasis: SPL7 dependent transcriptional regulation of cuproproteins, and PAA2/HMA8 turnover by the Clp system, which is independent on SPL7.

Prioritization of copper for the use in photosynthetic electron transport in developing leaves of hybrid poplar.

Plastocyanin (PC) is an essential and abundant copper (Cu) protein required for photosynthesis in higher plants. Severe copper deprivation has the potential to cause a defect in photosynthetic electron transport due to a lack in PC. The Cu-microRNAs, which are up-regulated under Cu deficiency, down-regulate the expression of target Cu proteins other than PC, cytochrome-c oxidase and the ethylene receptors. It has been proposed that this mechanism saves Cu for PC maturation. We aimed to test how hybrid poplar, a species that has capacity to rapidly expand its photosynthetically active tissue, responds to variations in Cu availability over time. Measurement of chlorophyll fluorescence after Cu depletion revealed a drastic effect on photosynthesis in hybrid poplar. The decrease in photosynthetic capacity was correlated with a reduction in PC protein levels. Compared to older leaves, PC decreased more strongly in developing leaves, which also lost more photosynthetic electron transport capacity. The effect of Cu depletion on older and more developed leaves was minor and these leaves maintained much of their photosynthetic capacity. Interestingly, upon resupply of Cu to the medium a very rapid recovery of Cu levels was seen in the younger leaves with a concomitant rise in the expression and activity of PC. In contrast, the expression of those Cu proteins, which are targets of microRNAs was under the same circumstances delayed. At the same time, Cu resupply had only minor effects on the older leaves. The data suggest a model where rapid recovery of photosynthetic capacity in younger leaves is made possible by a preferred allocation of Cu to PC in younger leaves, which is supported by Cu-microRNA expression.

Exploring the importance of sulfate transporters and ATP sulphurylases for selenium hyperaccumulation-a comparison of Stanleya pinnata and Brassica juncea (Brassicaceae).

Selenium (Se) hyperaccumulation, the capacity of some species to concentrate Se to levels upwards of 0.1% of dry weight, is an intriguing phenomenon that is only partially understood. Questions that remain to be answered are: do hyperaccumulators have one or more Se-specific transporters? How are these regulated by Se and sulfur (S)? In this study, hyperaccumulator Stanleya pinnata was compared with related non-hyperaccumulator Brassica juncea with respect to S-dependent selenate uptake and translocation, as well as for the expression levels of three sulfate/selenate transporters (Sultr) and three ATP sulphurylases (APS). Selenium accumulation went down ~10-fold with increasing sulfate supply in B. juncea, while S. pinnata only had a 2-3-fold difference in Se uptake between the highest (5 mM) and lowest sulfate (0 mM) treatments. The Se/S ratio was generally higher in the hyperaccumulator than the non-hyperaccumulator, and while tissue Se/S ratio in B. juncea largely reflected the ratio in the growth medium, S. pinnata enriched itself up to 5-fold with Se relative to S. The transcript levels of Sultr1;2 and 2;1 and APS1, 2, and 4 were generally much higher in S. pinnata than B. juncea, and the species showed differential transcript responses to S and Se supply. These results indicate that S. pinnata has at least one transporter with significant selenate specificity over sulfate. Also, the hyperaccumulator has elevated expression levels of several sulfate/selenate transporters and APS enzymes, which likely contribute to the Se hyperaccumulation and hypertolerance phenotype.

The Clp protease system is required for copper ion-dependent turnover of the PAA2/HMA8 copper transporter in chloroplasts.

The distribution of essential metal ions over subcellular compartments for use as cofactors requires control of membrane transporters. PAA2/HMA8 is a copper-transporting P1B -type ATPase in the thylakoid membrane, required for the maturation of plastocyanin. When copper is highly available to the plant this transporter is degraded, which implies the action of a protease. In order to identify the proteolytic machinery responsible for PAA2/HMA8 turnover in Arabidopsis, mutant lines defective in five different chloroplast protease systems were analyzed. Plants defective in the chloroplast caseinolytic protease (Clp) system were specifically impaired in PAA2/HMA8 protein turnover on media containing elevated copper concentrations. However, the abundance of a core Clp component was not directly affected by copper. Furthermore, the expression and activity of both cytosolic and chloroplast-localized superoxide dismutases (SODs), which are known to be dependent on copper, were not altered in the clp mutants, indicating that the loss of PAA2/HMA8 turnover in these lines was not caused by a lack of stromal copper. The results suggest that copper excess in the stroma triggers selection of the thylakoid-localized PAA2 transporter for degradation by the Clp protease, but not several other chloroplast proteases, and support a novel role for this proteolytic system in cellular copper homeostasis.

Copper Delivery to Chloroplast Proteins and its Regulation.

Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.

HMA1 and PAA1, two chloroplast-envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis.

Copper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA1.