Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficiency barley roots: a possible role for phytosiderophore production

To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 2.4.2.7) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1-sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed.     

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H₂O₂ concentration. In contrast, an early and high accumulation of H₂O₂ was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.     

Why are young rice plants highly susceptible to iron deficiency?

The reason why young rice plant is highly susceptible to Fe-deficiency was clarified as follows: Among Gramineae plants rice secreted a very low amount of deoxy-MA as a phytosiderophore even under Fe-deficiency, and the secretion by rice ceased within 10 days under Fe-deficiency although barley secreted MAs during a period of more than one month. When iron depletion continued, the rice root tips become chimeric and epidermal cells became necrotic. The mitochondrial membrane systems in the cortex cells were also severely damaged. Iron starvation occurred even in the mitochondria, and energy charge in the root decreased. This reduced energy charge has firstly diminished the secretion activity of deoxy-MA from the roots, secondly reduced the activity of the transporter which absorb deoxy-MA-Fe^III chelate and finally reduced the synthesis of deoxy-MA from metionine. Consequently, the depletion of Fe^II in the shoot was induced and severe chlorosis rapidly developed in the young rice plant under Fe-deficiency.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - MA mugineic acid - MAs mugineic acid-family phytosiderophores, it contains deoxy-MA, MA, epihydroxy-MA, hydroxy-MA, avenic acid and distichonic acid     

Cloning and characterization of purple acid phosphatase phytases from wheat, barley, maize, and rice

Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

胚乳对幼苗中苯丙氨酸解氨酶(PAL)活性的影响

在小麦、大麦、玉米黄化幼苗的萌发过程中,PAL活性有“消长”现象。当去胚乳后,幼苗中PAL活性普遍下降。亚胺环己酮处理可以阻止去胚乳造成的PAL活性下降。大麦、小麦去胚乳老化12h幼苗的提取液可在体外抑制整体苗的PAL活性。表明胚乳在调节幼苗体内PAL活性方面具有一定的作用。去胚乳后体内有PAL“抑制蛋白”的积累。     

Comparative mapping of the barley Ppd-H1 photoperiod response gene region,which lies close to a junction between two rice linkage segments

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.     

Interspecies compatibility of NAS1 gene promoters.

Nicotianamine and nicotianamine synthase (NAS) play key roles in iron nutrition in all higher plants. However, the mechanism underlying the regulation of NAS expression differs among plant species. Sequences homologous to iron deficiency-responsive elements (IDEs), i.e., cis-acting elements, are found on the promoters of these genes. We aimed to verify the interspecies compatibility of the Fe-deficiency response of NAS1 genes and understand the universal mechanisms that regulate their expression patterns in higher plants. Therefore, we introduced the graminaceous (Hordeum vulgare L. and Oryza sativa L.) NAS1 promoter::GUS into dicots (Nicotiana tabacum L. and Arabidopsis thaliana L.). Fe deficiency induced HvNAS1 expression in the shoots and roots when introduced into rice. HvNAS1 promoter::GUS and OsNAS1 promoter::GUS induced strong expression of GUS under Fe-deficient conditions in transformed tobacco. In contrast, these promoters only definitely functioned in Arabidopsis transformants. These results suggest that some Fe nutrition-related trans-factors are not compatible between graminaceous plants and Arabidopsis. HvNAS1 promoter::GUS induced GUS activity only in the roots of transformed tobacco under Fe-deficient conditions. On the other hand, OsNAS1 promoter::GUS induced GUS activity in both the roots and shoots of transformed tobacco under conditions of Fe deficiency. In tobacco transformants, the induction of GUS activity was induced earlier in the shoots than roots. These results suggest that the HvNAS1 and OsNAS1 promoters are compatible with Fe-acquisition-related trans-factors in the roots of tobacco and that the OsNAS1 promoter is also compatible with some shoot-specific Fe deficiency-related trans-factors in tobacco.     

Vector and Host-Plant Relationships of the Leafhopper-Borne Maize Yellow Stripe Virus

Maize yellow stripe virus (MYSV), associated with tenuivirus-like filaments, is transmitted in a persistent manner by the leafhopper Cicadulina chinai. In this vector, MYSV acquisition and inoculation threshold times were 30 min each, latent period ranged from 4.5 to 8 days depending on temperature (14-25 °C), and retention periods were as long as 27 days. Up to 26 % of C, chinai collected from maize fields in Giza, Egypt, during September and October 1985 were naturally infective with MYSV. Two symptom-types (fine and coarse stripe) appeared on experimentally infected plants, usually on separate leaves of the same plant. However, these two symptom-types could not be isolated on separate plants through transmission by single C. chinai leafhoppers. MYSV was transmitted by nymphs and adults of C. chinai from maize to maize, wheat and barley, and from wheat to maize plants. Up to 6 % of the wheat plants examined in Naga Hamadi (Southern Egypt) in February 1986, were naturally infected. It is suggested that wheat, barley and possibly graminaceous weeds may serve as winter hosts or reservoirs for MYSV and its leafhopper vector in Egypt.     

冬小麦植物铁载体分泌的杂种效应

缺铁是石灰性土壤常见的植物营养问题之一.禾本科植物种或基因型的植物铁载体分泌能力与耐缺铁有关,提高植物铁载体分泌能力是改良缺铁的土壤上植物铁aestivum L.) 3个杂交种及其4个亲本在缺铁营养液中植物铁载体的分泌及杂种的效应.植物铁载体的分泌率通过根分泌物对新形成的Fe(OH)3的活化能力进行测定, 在缺铁症出现时每隔2、3天测定1次.在缺铁条件下,所有基因型都分泌较多的植物铁载体,并且随缺铁症状的发展分泌量增加.杂交种具有对缺铁更敏感的反馈系统,在缺铁条件下,杂交种比亲本分泌铁载体的速度更快、量更高.通过分析杂交种和亲本的关系,认为可以通过对亲本分泌植物铁载体能力和配合力的选择,利用杂种优势来提高小麦铁的利用效率.     

Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes

One of the widest ranging abiotic stresses in world agriculture arises from low iron (Fe) availability due to high soil pH, with 30% of arable land too alkaline for optimal crop production. Rice is especially susceptible to low iron supply, whereas other graminaceous crops such as barley are not. A barley genomic DNA fragment containing two naat genes, which encode crucial enzymes involved in the biosynthesis of phytosiderophores, was introduced into rice using Agrobacterium-mediated transformation and pBIGRZ1. Phytosiderophores are natural iron chelators that graminaceous plants secrete from their roots to solubilize iron in the soil. The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants. Transgenic rice expressing the two genes showed a higher nicotianamine aminotransferase activity and secreted larger amounts of phytosiderophores than nontransformants under iron-deficient conditions. Consequently, the transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil.     

Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (Strategy II) in graminaceous plants

Nicotianamine aminotransferase (NAAT), the key enzyme involved in the biosynthesis of mugineic acid family phytosiderophores (MAs), catalyzes the amino transfer of nicotianamine (NA). MAs are found only in graminaceous plants, although NA has been detected in every plant so far investigated. Therefore, this amino transfer reaction is the first step in the unique biosynthesis of MAs that has evolved in graminaceous plants. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Based on the protein sequence of NAAT purified from Fe-deficient barley (Hordeum vulgare) roots, two distinct cDNA clones encoding NAAT, naat-A and naat-B, were identified. Their deduced amino acid sequences were homologous to several aminotransferases, and shared consensus sequences for the pyridoxal phosphate-binding site lysine residue and its surrounding residues. The expression of both naat-A and naat-B is increased in Fe-deficient barley roots, while naat-B has a low level of constitutive expression in Fe-sufficient barley roots. No detectable mRNA from either naat-A or naat-B was present in the leaves of either Fe-deficient or Fe-sufficient barley. One genomic clone with a tandem array of naat-B and naat-A in this order was identified. naat-B and naat-A each have six introns at the same locations. The isolation of NAAT genes will pave the way to understanding the mechanism of the response to Fe in graminaceous plants, and may lead to the development of cultivars tolerant to Fe deficiency that can grow in calcareous soils.     

Tolerance of crop plants to oxygen deficiency stress: fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia

The study investigates the reactions of rice, wheat and maize to anoxia (plants without access to oxygen) and hypoxia (roots with very limited access to oxygen). We studied the adaptations of these intact crop plants because they are known to differ widely in their tolerance to oxygen deficiency. In hypoxia, there was an accumulation of sugars, especially in wheat and maize, although both flood-sensitive species significantly increased the activities of fermentative and glycolytic enzymes, clearly more than in rice. In rice, avoiding an oxygen limitation due to the effective aeration system (30% of root cross-sectional area) may have accounted for only a minor metabolic reaction to hypoxia. In anoxia, maize and wheat quickly lost viability and nearly all photosynthetic capacity, while most rice leaves stayed turgid and green, losing only 50% of the photosynthetic capacity. A strong metabolic arrest under anoxia was obvious for the sucrolytic, glycolytic and fermentative enzymes in all tested species, but was most pronounced in rice. Of the 14 enzymes studied, rice showed the lowest activity increase in hypoxia for 11 enzymes, and the strongest activity decrease in anoxia for 8 enzymes. However, rice was able even under anoxia to keep a 1/4 of the ATP level of the aerated control, while it was at the detection limit in maize and wheat. It appears that in anoxic rice, the switch to metabolic dormancy and maintenance of basic shoot meristems diminishes the needs for energy and substrate. Additionally, rice already has lower sugar demand under hypoxia, and sugar supply appears to be sustained under anoxia by a functioning anaerobic amylase and by the photosynthetically active shoot.     

Iron acquisition by plants.

In nongraminaceous plants, the FeII-transporter gene and ferric-chelate reductase gene have been cloned from Arabidopsis thaliana, whereas FeIII-reductase has not. In graminaceous monocots, the genes for mugineic acids (MAs) synthesis, nas (nicotianamine synthase) and naat (nicotianamine aminotransferase), have been cloned from barley, whereas the FeIII-MAs transporter gene is yet to be cloned. Transferrin absorption in Dunaliella has been reported, suggesting a phagocytotic (endocytotic) Fe-acquisition mechanism. Work to develop transgenic cultivars tolerant to Fe-deficiency in calcareous soils is now in progress.     

Antioxidant defense in the leaves of C3 and C4 plants under salinity stress

The effect of salt stress (50, 100 and 150 m M of NaCl) on the activity of superoxide dismutase (SOD, EC. 1.15.1.1), ascorbate peroxidase (APX, EC. 1.11.1.11), glutathione reductase (GR, EC. 1.6.4.2) enzymes and also on the rate of lipid peroxidation in terms of thiobarbituric acid-reactive substances (TBARS) content and photosynthetic capacity in two wheat (C3 plants) and two maize (C4 plants) varieties was studied. In the non-salined control plants, the antioxidant enzymes activities were significantly higher for maize than for wheat. Adding salt to the nutrient solution increased the level of antioxidants in leaves of both maize and wheat. The first substantial response to salinity was found for SOD on the 2nd day, whereas changes occurred for APX on the 4th day and for GR on the 4th/5th day of salt treatment. Although SOD activity increased considerably more in wheat (C3), it never reached as high levels as in maize (C4) grown in the same treatment combinations. The total increase in APX activity was similar for wheat and maize, whereas GR activity was higher in leaves of maize. Lipid peroxidation analyses showed an increase in TBARS contents in both plants' species grown under salinity that corresponded to the damage that occurred in secondary oxidative stress. However, as a result of advanced antioxidant defense in maize, the TBARS quantities did not elevate to as high level as in wheat. Chlorophyll fluorescence measurements revealed a considerable decrease in the efficiency of PS II and electron-transport chain (ETC). Assimilation rate of CO₂ decreased in both plant groups; however, in C4 maize, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS. Results suggest that efficient antioxidant defense plays an important role in maize, the C₄ plant, resistance to environmental stresses like salinity or drought.     

Resistance gene analogs in barley and their relationship to rust resistance genes.

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.