OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds

Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium （Cd） in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling.     

Global Analysis of Gene Expression Profiles in Brassica napus Developing Seeds Reveals a Conserved Lipid Metabolism Regulation with Arabidopsis thaliana

In order to study Brassica napus fatty acid （FA） metabolism and relevant regulatory networks, a systematic identification of fatty acid （FA） biosynthesis-related genes was conducted. Following gene identification, gene expression profiles during B. napus seed development and FA metabolism were performed by cDNA chip hybridization （〉8000 EST clones from seed）. The results showed that FA biosynthesis and regulation, and carbon flux, were conserved between B. napus and Arabidopsis. However, a more critical role of starch metabolism was detected for B. napus seed FA metabolism and storage-component accumulation when compared with Arabidopsis. In addition, a crucial stage for the transition of seed-to-sink tissue was 17-21 d after flowering （DAF）, whereas FA biosynthesis-related genes were highly expressed pri- marily at 21 DAF. Hormone （auxin and jasmonate） signaling is found to be important for FA metabolism. This study helps to reveal the global regulatory network of FA metabolism in developing B. napus seeds.     

From Proteomics to Structural Studies of Cytosolic/MitochondriaI-Type Thioredoxin Systems in Barley Seeds

Thioredoxins （Trx） are ubiquitous proteins that participate in thiol disulfide reactions via two active site cysteine residues, allowing Trx to reduce disulfide bonds in target proteins. Recent progress in proteome analysis has resulted in identification of a wide range of potential target proteins for Trx, indicating that Trx plays a key role in several aspects of cell metabolism. In contrast to other organisms, plants contain multiple forms of Trx that are classified based on their primary structures and sub-cellular localization. The reduction of cytosolic and mitochondrial types of Trx is dependent on NADPH and catalyzed by NADPH-dependent thioredoxin reductase （NTR）. In barley, two isoforms each of Trx and NTR have been identified and investigated using proteomics, gene expression, and structural studies. This review outlines the diverse roles suggested for cytosolic/mitochondrial-type Trx systems in cereal seeds and summarizes the current knowledge of the barley system including recent data on function, regulation, interactions, and structure. Directions for future research are discussed.     

Characters of Cysteine Endopeptidases in Wheat Endosperm during Seed Germination and Subsequent Seedling Growth

The endopeptidases （EPs） in wheat endosperm during seed germination and subsequent seedling growth were characterized by gradient-polyacrylamide gel electrophoresis with gelatin copolymerized into the gel. Four cysteine EPs （EP1, EP2, EP3 and EP4） were detected in wheat endosperm during the 7 d growth after seed imbibition. The results also showed that the activities of all of these EPs increased continuously, and EP2 first appeared and had the highest proteolytic activity among the four EPs in this experimental process. The optimum pH and temperature of all four EPs were 4.0 and 40.0 ~C. All EPs were completely inhibited by 25 μmol/L E-64 and had no good thermal stabilities, especially EP1. In addition, these EPs had different substrate specificities to albumins, globulins, gliadins and glutenins; the main storage proteins of mature wheat endosperm. Among them, EP2 had the highest proteolytic activities on globulins, gliadins and glutenins, and might be the most important and specific EP with potential to be tightly correlated with seedling development.     

Shoot-Specific Down-Regulation of Protein Farnesyltransferase （α-Subunit） for Yield Protection against Drought in Canola

Canola （Brassica napus L.） is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid （ABA）, which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell＇s responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis β-subunit of farnesyltransferase （ERA1） enhances the plant＇s sensitivity to ABA and drought tolerance. Although the β-subunit of famesyltransferase （AtFTA） is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase （AtHPR1）, which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed thatAtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Arabidopsis RAN 1 Mediates Seed Development through Its Parental .Ratio by Affecting the Onset of Endosperm Cellularization

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 （RAN1） regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type （WT） and transgenic lines misexpressing RAN1 （msRAN1） gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 （MINI3）-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.     

Detection of Quantitative Trait Loci for Yield and Drought Tolerance Traits in Soybean Using a Recombinant Inbred Line Population

To investigate the genetic basis of drought tolerance in soybean （Glycine max L. Merr.） a recombinant inbred population with 184 F2：7：11 lines developed from a cross between Kefengl （drought tolerant） and Nannong1138-2 （drought sensitive） were tested under water-stressed and well-watered conditions in field and greenhouse trials. Traits measured included leaf wilting coefficient, excised leaf water loss and relative water content as indicators of plant water status and seed yield. A total of 40 quantitative trait loci （QTLs） were identified： 17 for leaf water status traits under drought stress and 23 for seed yield under well-watered and drought-stressed conditions in both field and greenhouse trials. Two seed yield QTLs were detected under both well-watered and drought-stressed conditions in the field on molecular linkage group H and Dlb, while two seed yield QTLs on molecular linkage group C2 were found under greenhouse conditions. Several QTLs for traits associated with plant water status were identified in both field and greenhouse trials, including two leaf wilting coefficient QTLs on molecular linkage group A2 and one excised leaf water loss QTL on molecular linkage group H. Phenotypic correlations of traits suggested several QTLs had pleiotropic or location-linked associations. These results will help to elucidate the genetic basis of drought tolerance in soybean, and could be incorporated into a marker-assisted selection breeding program to develop high-yielding soybean cultivars with improved tolerance to drought stress.     

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan I. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non- cellulosic polysaccharides of embryos. Arabinose levels decline to -15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee （Clausena lansium Skeels） axes and maize （Zea mays L.） embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase （SOD）, ascorbate peroxidase （APX） and catalase （CAT） of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

Anti-tumor activity and immunological modification of ribosome-inactivating protein （RIP） from Momordica charantia by covalent attachment of polyethylene glycol

Ribosome-inactivating proteins （RIPs） are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosisinducing activity, and polyethylene glycol （PEG） modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of N- Asp-Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the acti- vation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa （mPEG）2-Lys-NHS. The inhibition activity of both PEGylated and non- PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.     

Oxygation Enhances Growth,Gas Exchange and Salt Tolerance of Vegetable Soybean and Cotton in a Saline Vertisol

Impacts of salinity become severe when the soil is deficient in oxygen. OxygaUon （using aerated water for subsurface drip irrigation of crop） could minimize the impact of salinity on plants under oxygen-limiting soil environments. Pot experiments were conducted to evaluate the effects of oxygation （12% air volume/volume of water） on vegetable soybean （moderately salt tolerant） and cotton （salt tolerant） in a salinized vertisol at 2, 8, 14, 20 dS/m ECe. In vegetable soybean, oxygation increased above ground biomass yield and water use efficiency （WUE） by 13% and 22%, respectively, compared with the control. Higher yield with oxygation was accompanied by greater plant height and stem diameter and reduced specific leaf area and leaf Na^＋ and CI^- concentrations. In cotton, oxygation increased lint yield and WUE by 18% and 16%, respectively, compared with the control, and was accompanied by greater canopy light interception, plant height and stem diameter. Oxygation also led to a greater rate of photosynthesis, higher relative water content in the leaf, reduced crop water stress index and lower leaf water potential. It did not, however, affect leaf Na^＋ or CI^- concentration. Oxygation invariably increased, whereas salinity reduced the K^＋： Na^＋ ratio in the leaves of both species. Oxygation improved yield and WUE performance of salt tolerant and moderately tolerant crops under saline soil environments, and this may have a significant impact for irrigated agriculture where saline soils pose constraints to crop production.     

Vegetation Mapping of the Mond Protected Area of Bushehr Province （South-west Iran）

Arid regions of the world occupy up to 35% of the earth＇s surface, the basis of various definitions of climatic conditions, vegetation types or potential for food production. Due to their high ecological value, monitoring of arid regions is necessary and modern vegetation studies can help in the conservation and management of these areas. The use of remote sensing for mapping of desert vegetation is difficult due to mixing of the spectral reflectance of bright desert soils with the weak spectral response of sparse vegetation. We studied the vegetation types in the semiarid to arid region of Mond Protected Area, south-west Iran, based on unsupervised classification of the Spot XS bands and then produced updated maps. Sixteen map units covering 12 vegetation types were recognized in the area based on both field works and satellite mapping. Halocnemum strobilaceum and Suaeda fruticosa vegetation types were the dominant types and Ephedra foliata, Salicornia europaea-Suaeda heterophylla vegetation types were the smallest. Vegetation coverage decreased sharply with the increase in salinity towards the coastal areas of the Persian Gulf. The highest vegetation coverage belonged to the riparian vegetation along the Mond River, which represents the northern boundary of the protected area. The location of vegetation types was studied on the separate soil and habitat diversity maps of the study area, which helped in final refinements of the vegetation map produced.     

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Induction of the AOX1D Isoform of Alternative Oxidase in A. thaliana T-DNA Insertion Lines Lacking Isoform AOX1A Is Insufficient to Optimize Photosynthesis when Treated with Antimycin A

Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.