Enhanced expression of OsNAC5 leads to up-regulation of OsNAC6 and changes rice (Oryza sativa L.) ionome.

NAC transcription factors are plant-specific proteins involved in many processes during the plant life cycle and responses to biotic and abiotic stresses. Previous studies have shown that stress-induced OsNAC5 from rice (Oryza sativa L.) is up-regulated by senescence and might be involved in control of iron (Fe) and zinc (Zn) concentrations in rice seeds. Aiming a better understanding of the role of OsNAC5 in rice plants, we investigated a mutant line carrying a T-DNA insertion in the promoter of OsNAC5, which resulted in enhanced expression of the transcription factor. Plants with OsNAC5 enhanced expression were shorter at the seedling stage and had reduced yield at maturity. In addition, we evaluated the expression level of OsNAC6, which is co-expressed with OsNAC5, and found that enhanced expression of OsNAC5 leads to increased expression of OsNAC6, suggesting that OsNAC5 might regulate OsNAC6 expression. Ionomic analysis of leaves and seeds from the OsNAC5 enhanced expression line revealed lower Fe and Zn concentrations in leaves and higher Fe concentrations in seeds than in WT plants, further suggesting that OsNAC5 may be involved in regulating the ionome in rice plants. Our work shows that fine-tuning of transcription factors is key when aiming at crop improvement.

Chromosomal introgressions from Oryza meridionalis into domesticated rice Oryza sativa result in iron tolerance.

Iron (Fe) toxicity is one of the most common mineral disorders affecting rice (Oryza sativa) production in flooded lowland fields. Oryza meridionalis is indigenous to northern Australia and grows in regions with Fe-rich soils, making it a candidate for use in adaptive breeding. With the aim of understanding tolerance mechanisms in rice, we screened a population of interspecific introgression lines from a cross between O. sativa and O. meridionalis for the identification of quantitative trait loci (QTLs) contributing to Fe-toxicity tolerance. Six putative QTLs were identified. A line carrying one introgression from O. meridionalis on chromosome 9 associated with one QTL was highly tolerant despite very high shoot Fe concentrations. Physiological, biochemical, ionomic, and transcriptomic analyses showed that the tolerance of the introgression lines could partly be explained by higher relative Fe retention in the leaf sheath and culm. We constructed the interspecific hybrid genome in silico for transcriptomic analysis and identified differentially regulated introgressed genes from O. meridionalis that could be involved in shoot-based Fe tolerance, such as metallothioneins, glutathione S-transferases, and transporters from the ABC and MFS families. This work demonstrates that introgressions of O. meridionalis into the O. sativa genome can confer increased tolerance to excess Fe.

The copper economy response is partially conserved in rice (Oryza sativa L.).

Copper (Cu) is an essential element for plants, especially in photosynthesis, as it is required for plastocyanin function in electron transfer reactions at thylakoid membranes. In Arabidopsis thaliana, Cu deficiency leads to the Cu economy response, in which plants prioritize Cu usage by plastocyanin in detriment of non-essential cupric proteins. In rice (Oryza sativa), however, this response has not been characterized. Rice OsHMA5 is a Cu xylem-loading transporter involved in Cu translocation from roots to shoots, as suggested by the analysis of oshma5 mutant plants. Aiming to understand how rice plants respond to Cu deficiency and how decreased Cu translocation to shoots can affect this response, we characterized the physiological and molecular responses of WT and oshma5 plants under control and Cu deficiency treatments. We found evidence that shoots of oshma5 plants are more prone to Cu deficiency compared to shoots of WT plants, as demonstrated by decreased chlorophyll and Cu concentrations, and electron transport rate. Gene expression analysis revealed that Cu high-affinity transporters OsCOPT1 and OsCOPT5, along with a set of miRNAs and three Cu/Zn superoxide dismutases are responsive to Cu deficiency in both WT and oshma5 plants, suggesting their involvement in the Cu economy response. However, Fe superoxide dismutase was not up-regulated in rice, indicating a difference compared to the A. thaliana Cu economy model. Therefore, we provide evidence for a partially conserved Cu economy response in rice, in comparison to A. thaliana.

Root responses of contrasting rice genotypes to low temperature stress.

Rice (Oryza sativa L.) ssp. indica is the most cultivated species in the South of Brazil. However, these plants face low temperature stress from September to November, which is the period of early sowing, affecting plant development during the initial stages of growth, and reducing rice productivity. This study aimed to characterize the root response to low temperature stress during the early vegetative stage of two rice genotypes contrasting in their cold tolerance (CT, cold-tolerant; and CS, cold-sensitive). Root dry weight and length, as well as the number of root hairs, were higher in CT than CS when exposed to cold treatment. Histochemical analyses indicated that roots of CS genotype present higher levels of lipid peroxidation and H2O2 accumulation, along with lower levels of plasma membrane integrity than CT under low temperature stress. RNAseq analyses revealed that the contrasting genotypes present completely different molecular responses to cold stress. The number of over-represented functional categories was lower in CT than CS under cold condition, suggesting that CS genotype is more impacted by low temperature stress than CT. Several genes might contribute to rice cold tolerance, including the ones related with cell wall remodeling, cytoskeleton and growth, signaling, antioxidant system, lipid metabolism, and stress response. On the other hand, high expression of the genes SRC2 (defense), root architecture associated 1 (growth), ACC oxidase, ethylene-responsive transcription factor, and cytokinin-O-glucosyltransferase 2 (hormone-related) seems to be related with cold sensibility. Since these two genotypes have a similar genetic background (sister lines), the differentially expressed genes found here can be considered candidate genes for cold tolerance and could be used in future biotechnological approaches aiming to increase rice tolerance to low temperature.

Physiological responses of rice (Oryza sativa L.) oszip7 loss-of-function plants exposed to varying Zn concentrations.

Rice is a daily staple for half of the world's population. However, rice grains are poor in micronutrients such as Fe and Zn, the two most commonly deficient minerals in the human diet. In plants, Fe and Zn must be absorbed from the soil, distributed and stored, so that their concentrations are maintained at sufficient but non-toxic levels. The understanding of mechanisms of Fe and Zn homeostasis in plants has the potential to benefit agriculture, improving the use of micronutrients by plants, as well as to indicate approaches that aim at biofortification of the grains. ZIP transporters are commonly associated with Zn uptake, but there are few reports about their physiological relevance in planta. Here we describe a Tos17 loss-of-function line for the Zn plasma membrane transporter OsZIP7 (oszip7). We showed that the absence of functional OsZIP7 leads to deregulated Zn partitioning, increasing Zn accumulation in roots but decreasing in shoots and seeds. We also demonstrated that, upon Zn deficiency, oszip7 plants slightly increase their photosynthetic performance, suggesting that these plants might be primed for Zn deficiency which makes them more tolerant. On the other hand, we found that Zn excess is more deleterious to oszip7 plants compared to wild type, which may be linked to secondary effects in concentrations of other elements such as Fe. Our data suggest that OsZIP7 is important for Zn homeostasis under physiological Zn concentrations, and that Fe homeostasis might be affected due to loss of function of OsZIP7.

Photosynthetic activity of indica rice sister lines with contrasting cold tolerance.

Incidence of cold during early stages is an obstacle for the growing progress of rice plants. Cold stress has strong negative effects on photosynthetic activity. Previously, our group evaluated plant survival of 90 indica rice genotypes after cold treatment. Two sister lines were characterized as cold-tolerant and cold-sensitive. Transcriptomic analyses of the same genotypes had indicated differential expression of genes related to photosynthesis. Previous work with japonica rice had suggested that cold sensitivity was more related to photosystem II (PSII) than to photosystem I (PSI). Using our previously identified contrasting genotypes, we investigated the role of specific steps of the photosynthetic process in cold tolerance/sensitivity of indica rice plants during and after (recovery period) cold exposure. During both cold treatment and recovery period, the photochemical activity (including PSII and PSI) presented higher levels in the low temperature-tolerant genotype, when compared with the sensitive one. The higher photochemical efficiency during the cold treatment appears to be related to a lower fraction of reduced QA - in PSII. We also observed lower transpiration rates and higher water use efficiency in the cold-tolerant genotype, due to stomatal closure. After the recovery period, the higher efficiency in the cold-tolerant genotype seems to be related to a lower fraction of reduced QA - and a larger pool of final electron acceptors at the PSI. This work uncovered changes in photosynthetic performance including both photosystems and improved water use efficiency which may be important components of cold tolerance mechanisms in indica rice.

The Mitochondrial Iron-Regulated (MIR) gene is Oryza genus specific and evolved before speciation within the Oryza sativa complex.

MAIN CONCLUSION: The MIR gene is not an Oryza sativa orphan gene, but an Oryza genus-specific gene that evolved before AA lineage speciation by a complex origination process. Rice (Oryza sativa L.) is a model species and an economically relevant crop. The Oryza genus comprises 25 species, with genomic data available for several Oryza species, making it a model for genetics and evolution. The Mitochondrial Iron-Regulated (MIR) gene was previously implicated in the O. sativa Fe deficiency response, and was considered an orphan gene present only in rice. Here we show that MIR is also found in other Oryza species that belong to the Oryza sativa complex, which have AA genome type and constitute the primary gene pool for O. sativa breeding. Our data suggest that MIR originated in a stepwise process, in which sequences derived from an exon fragment of the raffinose synthase gene were pseudogenized into non-coding, which in turn originated the MIR gene de novo. All species with a putative functional MIR gene conserve their regulation by Fe deficiency, with the exception of Oryza barthii. In O. barthii, the MIR coding sequence was translocated to a different chromosomal position and separated from its regulatory region, leading to a lack of Fe deficiency responsiveness. Moreover, the MIR co-expression subnetwork cluster in O. sativa is responsive to Fe deficiency, evidencing the importance of the newly originated gene in Fe uptake. This work establishes that MIR is not an orphan gene as previously proposed, but a de novo originated gene within the genus Oryza. We also showed that MIR is undergoing genomic changes in one species (O. barthii), with an impact on Fe deficiency response.

The Combined Strategy for iron uptake is not exclusive to domesticated rice (Oryza sativa).

Iron (Fe) is an essential micronutrient that is frequently inaccessible to plants. Rice (Oryza sativa L.) plants employ the Combined Strategy for Fe uptake, which is composed by all features of Strategy II, common to all Poaceae species, and some features of Strategy I, common to non-Poaceae species. To understand the evolution of Fe uptake mechanisms, we analyzed the root transcriptomic response to Fe deficiency in O. sativa and its wild progenitor O. rufipogon. We identified 622 and 2,017 differentially expressed genes in O. sativa and O. rufipogon, respectively. Among the genes up-regulated in both species, we found Fe transporters associated with Strategy I, such as IRT1, IRT2 and NRAMP1; and genes associated with Strategy II, such as YSL15 and IRO2. In order to evaluate the conservation of these Strategies among other Poaceae, we identified the orthologs of these genes in nine species from the Oryza genus, maize and sorghum, and evaluated their expression profile in response to low Fe condition. Our results indicate that the Combined Strategy is not specific to O. sativa as previously proposed, but also present in species of the Oryza genus closely related to domesticated rice, and originated around the same time the AA genome lineage within Oryza diversified. Therefore, adaptation to Fe2+ acquisition via IRT1 in flooded soils precedes O. sativa domestication.

Genotype Variation in Rice (Oryza sativa L.) Tolerance to Fe Toxicity Might Be Linked to Root Cell Wall Lignification.

Iron (Fe) is an essential element to plants, but can be harmful if accumulated to toxic concentrations. Fe toxicity can be a major nutritional disorder in rice (Oryza sativa) when cultivated under waterlogged conditions, as a result of excessive Fe solubilization of in the soil. However, little is known about the basis of Fe toxicity and tolerance at both physiological and molecular level. To identify mechanisms and potential candidate genes for Fe tolerance in rice, we comparatively analyzed the effects of excess Fe on two cultivars with distinct tolerance to Fe toxicity, EPAGRI 108 (tolerant) and BR-IRGA 409 (susceptible). After excess Fe treatment, BR-IRGA 409 plants showed reduced biomass and photosynthetic parameters, compared to EPAGRI 108. EPAGRI 108 plants accumulated lower amounts of Fe in both shoots and roots compared to BR-IRGA 409. We conducted transcriptomic analyses of roots from susceptible and tolerant plants under control and excess Fe conditions. We found 423 up-regulated and 92 down-regulated genes in the susceptible cultivar, and 42 up-regulated and 305 down-regulated genes in the tolerant one. We observed striking differences in root gene expression profiles following exposure to excess Fe: the two cultivars showed no genes regulated in the same way (up or down in both), and 264 genes were oppositely regulated in both cultivars. Plants from the susceptible cultivar showed down-regulation of known Fe uptake-related genes, indicating that plants are actively decreasing Fe acquisition. On the other hand, plants from the tolerant cultivar showed up-regulation of genes involved in root cell wall biosynthesis and lignification. We confirmed that the tolerant cultivar has increased lignification in the outer layers of the cortex and in the vascular bundle compared to the susceptible cultivar, suggesting that the capacity to avoid excessive Fe uptake could rely in root cell wall remodeling. Moreover, we showed that increased lignin concentrations in roots might be linked to Fe tolerance in other rice cultivars, suggesting that a similar mechanism might operate in multiple genotypes. Our results indicate that changes in root cell wall and Fe permeability might be related to Fe toxicity tolerance in rice natural variation.

Deep RNAseq indicates protective mechanisms of cold-tolerant indica rice plants during early vegetative stage.

KEY MESSAGE: Cold-tolerance in rice may be related to increased cellulose deposition in the cell wall, membrane fatty acids unsaturation and differential expression of several newly identified genes. Low temperature exposure during early vegetative stages limits rice plant's growth and development. Most genes previously related to cold tolerance in rice are from the japonica subspecies. To help clarify the mechanisms that regulate cold tolerance in young indica rice plants, comparative transcriptome analysis of 6 h cold-treated (10 °C) leaves from two genotypes, cold-tolerant (CT) and cold-sensitive (CS), was performed. Differentially expressed genes were identified: 831 and 357 sequences more expressed in the tolerant and in the sensitive genotype, respectively. The genes with higher expression in the CT genotype were used in systems biology analyses to identify protein-protein interaction (PPI) networks and nodes (proteins) that are hubs and bottlenecks in the PPI. From the genes more expressed in the tolerant plants, 60% were reported as affected by cold in previous transcriptome experiments and 27% are located within QTLs related to cold tolerance during the vegetative stage. Novel cold-responsive genes were identified. Quantitative RT-PCR confirmed the high-quality of RNAseq libraries. Several genes related to cell wall assembly or reinforcement are cold-induced or constitutively highly expressed in the tolerant genotype. Cold-tolerant plants have increased cellulose deposition under cold. Genes related to lipid metabolism are more expressed in the tolerant genotype, which has higher membrane fatty acids unsaturation, with increasing levels of linoleic acid under cold. The CT genotype seems to have higher photosynthetic efficiency and antioxidant capacity, as well as more effective ethylene, Ca2+ and hormone signaling than the CS. These genes could be useful in future biotechnological approaches aiming to increase cold tolerance in rice.

OsMTP11 is localised at the Golgi and contributes to Mn tolerance.

Membrane transporters play a key role in obtaining sufficient quantities of manganese (Mn) but also in protecting against Mn toxicity. We have characterized OsMTP11, a member of the Cation Diffusion Facilitator/Metal Tolerance Protein (CDF/MTP) family of metal cation transporters in Oryza sativa. We demonstrate that OsMTP11 functions in alleviating Mn toxicity as its expression can rescue the Mn-sensitive phenotype of the Arabidopsis mtp11-3 knockout mutant. When expressed stably in Arabidopsis and transiently in rice and tobacco, it localises to the Golgi. OsMTP11 partially rescues the Mn-hypersensitivity of the pmr1 yeast mutant but only slightly alleviates the Zn sensitivity of the zrc1 cot1 yeast mutant. Overall, these results suggest that OsMTP11 predominantly functions as a Mn-transporting CDF with lower affinity for Zn. Site-directed mutagenesis studies revealed four substitutions in OsMTP11 that appear to alter its transport activity. OsMTP11 harbouring a substitution of leucine 150 to a serine fully rescued pmr1 Mn-sensitivity at all concentrations tested. The other substitutions, including those at conserved DxxxD domains, reduced complementation of pmr1 to different levels. This indicates their importance for OsMTP11 function and is a starting point for refining transporter activity/specificity.

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications.

Zinc (Zn) is an essential micronutrient for all organisms, with key catalytic and structural functions. Zn deficiency in plants, common in alkaline soils, results in growth arrest and sterility. On the other hand, Zn can become toxic at elevated concentrations. Several studies revealed molecules involved with metal acquisition in roots, distribution within the plant and translocation to seeds. Transmembrane Zn transport proteins and Zn chelators are involved in avoiding its toxic effects. Plant species with the capacity to hyperaccumulate and hypertolerate Zn have been characterized. Plants that accumulate and tolerate high amounts of Zn and produce abundant biomass may be useful for phytoremediation, allowing cleaning of metal-contaminated soils. The study of Zn hyperaccumulators may provide indications of genes and processes useful for biofortification, for developing crops with high amounts of nutrients in edible tissues. Future research needs to focus on functional characterization of Zn transporters in planta, elucidation of Zn uptake and sensing mechanisms, and on understanding the cross-talk between Zn homeostasis and other physiological processes. For this, new research should use multidisciplinary approaches, combining traditional and emerging techniques, such as genome-encoded metal sensors and multi-element imaging, quantification and speciation using synchrotron-based methods.

Functional analysis of the rice vacuolar zinc transporter OsMTP1.

Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation.

Iron biofortification in rice: it's a long way to the top.

Rice and most staple cereals contain low iron (Fe) levels, most of which is lost during grain processing. Populations with monotonous diets consisting mainly of cereals are especially prone to Fe deficiency, which affects about two billion people. Supplementation or food fortification programs have not always been successful. Crop Fe fertilization is also not very effective due to Fe soil insolubility. An alternative solution is Fe biofortification by generating cultivars that efficiently mobilize, uptake and translocate Fe to the edible parts. Here, we review the strategies used for the Fe biofortification of rice, including conventional breeding and directed genetic modification, which offer the most rapid way to develop Fe-rich rice plants. While classical breeding is able to modify the contents of inhibitors of Fe absorption, transgenic approaches have focused on enhanced Fe uptake from soil, xylem and phloem loading and grain sink strength. A comprehensive table is provided in which the percentages of the recommended dietary Fe intake reached by independently developed transgenic plants are calculated. In this review we also emphasize that the discovery of new QTLs and genes related to Fe biofortification is extremely important, but interdisciplinary research is needed for future success in this area.

Effects of different Fe supplies on mineral partitioning and remobilization during the reproductive development of rice (Oryza sativa L.).

BACKGROUND: Minimal information exists on whole-plant dynamics of mineral flow through rice plants and on the source tissues responsible for mineral export to developing seeds. Understanding these phenomena in a model plant could help in the development of nutritionally enhanced crop cultivars. A whole-plant accumulation study, using harvests during reproductive development under different Fe supplies, was conducted to characterize mineral accumulation in roots, non-flag leaves, flag leaves, stems/sheaths, and panicles of Kitaake rice plants. RESULTS: Low Fe supply promoted higher accumulation of Zn, Cu and Ni in roots, Mn, Ca, Mg and K in leaves and Zn in stems/sheaths and a smaller accumulation of Fe, Mn and Ca in roots and Zn and Ni in leaves. High Fe supply promoted higher accumulation of Fe in roots and Zn in leaves and a smaller accumulation of Fe in leaves and stems/sheaths and Zn, Cu and K in roots. Correlation analyzes indicated that fluctuations in Mn-Ca, Zn-Cu, Zn-Ni, Cu-Ni, Mo-S, Ca-Mg, Cu-Mn and Cu-Mg concentrations in response to different Fe supplies were positively correlated in at least four of the five organs analyzed. CONCLUSIONS: Mineral content loss analysis indicated that mineral remobilization from vegetative organs can occur in rice plants; however, for seeds to acquire minerals, vegetative remobilization is not absolutely required. Also, mineral remobilization from vegetative tissues in rice was greatly dependent of plant Fe nutrition. Remobilization was observed for several minerals from flag leaves and stems/sheaths, but the amounts were generally far below the total mineral accretion observed in panicles, suggesting that continued uptake and translocation of minerals from the roots during seed fill are probably more important than mineral remobilization.

Identification of Fe-excess-induced genes in rice shoots reveals a WRKY transcription factor responsive to Fe, drought and senescence.

Fe participates in several important reactions in plant metabolism. However, Fe homeostasis in plants is not completely understood, and molecular studies on Fe-excess stress are scarce. Rice (Oryza sativa L. ssp. indica) is largely cultivated in submerged conditions, where the extremely reductive environment can lead to severe Fe overload. In this work, we used representational difference analysis (RDA) to isolate sequences up-regulated in rice shoots after exposure to Fe-excess. We isolated 24 sequences which have putative functions in distinct cellular processes, such as transcription regulation (OsWRKY80), stress response (OsGAP1, DEAD-BOX RNA helicase), proteolysis (oryzain-α, rhomboid protein), photosynthesis (chlorophyll a/b binding protein), sugar metabolism (ß glucosidase) and electron transport (NADH ubiquinone oxireductase). We show that the putative WRKY transcription factor OsWRKY80 is up-regulated in rice leaves, stems and roots after Fe-excess treatment. This up-regulation is also observed after dark-induced senescence and drought stress, indicating that OsWRKY80 could be a general stress-responsive gene. To our knowledge, this is the first report of an Fe-excess-induced transcription factor in plants.

Identification of up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent transcription factor.

Rice is a poor source of micronutrients such as iron and zinc. To help clarify the molecular mechanisms that regulate metal mobilization from leaves to developing seeds, we conducted suppression subtractive hybridization analysis in flag leaves of two rice cultivars. Flag leaves are the major source of remobilized metals for developing seeds. We isolated 78 sequences up-regulated in flag leaves at the grain filling stage relative to the panicle exertion stage. Differential expression of selected genes (encoding 7 transport proteins, the OsNAS3 enzyme and the OsNAC5 transcription factor) was confirmed by quantitative RT-PCR. We show that OsNAC5 expression is up-regulated by natural (aging) and induced senescence processes (dark, ABA application, high salinity, cold and Fe-deficiency) and its expression is not affected in the presence of 6-benzylaminopurine (a senescence inhibitor) under dark-induced senescence. Salt induction of OsNAC5 expression is abolished by nicotinamide, an inhibitor of ABA effects. This result and the presence of cis-acting elements in the promoter region of the OsNAC5 gene suggest an ABA-dependent regulation. Using four different rice cultivars, we show that OsNAC5 up-regulation is higher and earlier in flag leaves and panicles of IR75862 plants, which have higher seed concentrations of Fe, Zn and protein. We suggest that OsNAC5 is a novel senescence-associated ABA-dependent NAC transcription factor and its function could be related to Fe, Zn and amino acids remobilization from green tissues to seeds.

Increased senescence-associated gene expression and lipid peroxidation induced by iron deficiency in rice roots.

Iron deficiency is among the most common nutritional disorders in plants. Low iron supply causes decreased root growth and even plant death. However, there are no reports about the specific pathways that lead Fe-deficient roots to senescence and death. To investigate the molecular mechanisms that regulate rice roots response to Fe-deficiency, rice seedlings were grown for 3, 6 and 9 days in the presence or absence of Fe. Sequences of 28 induced genes in rice roots under Fe-deficiency were identified by representational difference analysis (RDA). About 40% of these sequences have been previously reported as senescence-related. Differential expression of selected genes was confirmed by semi-quantitative RT-PCR analysis. Classical senescence-related sequences, such as MYB and WRKY transcription factors, cysteine protease, ubiquitin-conjugating enzyme, lipid transfer protein, fatty acid hydroxylase, beta-glucosidase and cytochrome P450 oxydoreductase were identified. Fe-deficiency also resulted in decreased dry weight, increased lipid peroxidation (detected by TBA and histochemical methods) as well as evident membrane damage in Fe-deficient roots. Taken together, the results indicate that Fe-deficiency in roots is linked to typical senescence pathways, associated with lipid peroxidation.

Iron deficiency in rice shoots: identification of novel induced genes using RDA and possible relation to leaf senescence.

Rice plants are highly susceptible to Fe-deficiency. Under nutrient deprivation, plant cells undergo extensive metabolic changes for their continued survival. To provide further insight into the pathways induced during Fe-deficiency, rice seedlings were grown for 3, 6 and 9 days in the presence or absence of Fe. Using RDA (Representational Difference Analysis), sequences of 32 induced genes in rice shoots under Fe-deficiency were identified. About 30% of the sequences found have been previously reported as responsive to other abiotic and even biotic stresses. However, this is the first report that indicates their relation to Fe deprivation. Differential expression of selected genes was confirmed by semi-quantitative RT-PCR analysis. The identification of classical senescence-related sequences, such as lipase EC 3.1.1.-, ubiquitin-conjugating enzyme EC 6.3.2.19, beta-Glucosidase EC 3.2.1.21 and cysteine synthase EC 2.5.1.47, besides the higher accumulation of total soluble sugars prior to the decrease of total chlorophyll content in Fe-deficient leaves, indicate that sugar accumulation may be one of the factors leading to premature leaf senescence induced by Fe-deficiency.

Environmental and ontogenetic control of accumulation of brachycerine, a bioactive indole alkaloid from Psychotria brachyceras.

Brachycerine is a monoterpenoid indole alkaloid accumulated in Psychotria brachyceras plants (Rubiaceae). To better understand the accumulation patterns of this alkaloid, we investigated its content in different plant organs from field-grown trees, throughout the seasons, during seedling development, and in response to potential biotic factors regulating its biosynthesis. Quantification by RP-HPLC showed that aerial vegetative organs (green stems, young and old leaves) yielded similar amounts of brachycerine [0.1-0.2% dry weight (DW)]. Brachycerine was not detected in roots. In reproductive structures, the highest brachycerine amounts (0.3% DW) were found in inflorescences. Alkaloid concentration decreased in mature fruits (0.045% DW). The lowest concentration in reproductive organs was observed in quiescent seeds (0.004% DW). Apparently, brachycerine content dropped during radicle emission in germinating seeds. During seedling development, an increase in leaf content from 0.02 to 0.1% DW was observed between the stages of 2 and 14 leaves, respectively. Salicylic acid did not affect brachycerine content. A doubling of alkaloid content was observed in wounded plants, and a threefold induction occurred with jasmonic acid treatment, suggesting that brachycerine biosynthesis is regulated by jasmonate production.