Abscisic acid cross-talking with hydrogen peroxide and osmolyte compounds may regulate the leaf rolling mechanism under drought

Leaf rolling observed in some crops such as maize, rice, wheat and sorghum is an indicator of decreased water status. Moderate leaf rolling not tightly or early increases the photosynthesis and grain yield of crop cultivars under environmental stresses. Moreover, the effects of exogenous abscisic acid (ABA) on stomatal conductance, water status and synthesis of osmotic compounds are a well-known issue in plants subjected to water deficit. However, it is not clear how the cross-talk of ABA with H₂O₂ and osmolyte compounds affects the leaf rolling mechanism. Regulation mechanism of leaf rolling by ABA has been first studied in maize seedlings under drought stress induced by polyethylene glycol 6000 (PEG 6000) in this study. ABA treatment under drought stress reduced hydrogen peroxide (H₂O₂) content and the degree of leaf rolling (%) while the treatment-induced ABA synthesis, osmolyte levels (proline, polyamine and total soluble sugars) and some antioxidant enzyme activities in comparison to the plants that were not treated with ABA. Furthermore, exogenous ABA up-regulated the expression levels of arginine decarboxylase (ADC) and pyrroline-5-carboxylate synthase (P5CS) genes and down-regulated polyamine oxidase (PAO), diamine oxidase (DAO) and proline dehydrogenase (ProDH) gene expressions. When endogenous ABA content was decreased by the treatment of fluoridone (FLU) that is an ABA inhibitor, leaf rolling degree (%), H₂O₂ content and antioxidant enzyme activities increased, but osmolyte levels, ADC and P5CS gene expressions decreased. Finally, the treatment of ABA to maize seedlings exposed to drought stress resulted in the stimulation of the antioxidant system, osmotic adjustment and reduction of leaf rolling. We concluded that ABA can be a signal compound cross-talking H₂O₂, proline and polyamines and thus involved in the leaf rolling mechanism by providing osmotic adjustment. The results of this study can be used to provide data for the molecular breeding of maize hybrids with high grain yield by means of moderately rolled leaves.     

Comparative Proteomic Analysis of Differentially Expressed Proteins Induced by Hydrogen Sulfide in Spinacia oleracea Leaves

Hydrogen sulfide (H₂S), as a potential gaseous messenger molecule, has been suggested to play important roles in a wide range of physiological processes in plants. The aim of present study was to investigate which set of proteins is involved in H₂S-regulated metabolism or signaling pathways. Spinacia oleracea seedlings were treated with 100 µM NaHS, a donor of H₂S. Changes in protein expression profiles were analyzed by 2-D gel electrophoresis coupled with MALDI-TOF MS. Over 1000 protein spots were reproducibly resolved, of which the abundance of 92 spots was changed by at least 2-fold (sixty-five were up-regulated, whereas 27 were down-regulated). These proteins were functionally divided into 9 groups, including energy production and photosynthesis, cell rescue, development and cell defense, substance metabolism, protein synthesis and folding, cellular signal transduction. Further, we found that these proteins were mainly localized in cell wall, plasma membrane, chloroplast, mitochondria, nucleus, peroxisome and cytosol. Our results demonstrate that H₂S is involved in various cellular and physiological activities and has a distinct influence on photosynthesis, cell defense and cellular signal transduction in S. oleracea leaves. These findings provide new insights into proteomic responses in plants under physiological levels of H₂S.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.     

Regulatory function of AMP1 in ABA biosynthesis and drought resistance in arabidopsis

In this study we reported the isolation of a mutant in which the reporter pVP14-LUC was highly expressed in Arabidopsis. The gene expression of maize VP14 is closely correlated with the endogenous ABA levels, and the Arabidopsis homolog of VP14, AtNCED1, encoding an enzyme of ABA biosynthesis, was up-regulated, and high ABA level was detected in the mutant. Map-based cloning revealed that the mutated gene is a novel allele of the AMP1 (Altered Meristem Program 1) which encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We found that the mutant displayed obvious drought tolerance, being with more lateral roots, high seed germination under mannitol, increased ABA accumulation, and highly induced gene expression of RD29A. Using the approaches of artificial microRNA gene silencing in transgenic plants, three AMP1 down-regulated lines were obtained. The AMP1 down-regulated plants exhibited a low rate of water loss, decreased stomatal aperture, and enhanced drought tolerance. These results provide evidence demonstrating the regulatory function of AMP1 in plant drought tolerance and stress responsive gene expression.     

The maize Viviparous10/Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesis

Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele of vp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABA-biosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses.     

Changes in Phosphorylation Status of Wheat Plastid Polypeptides as Influenced by Light, Calcium and Cyclic AMP

The polypeptides of etioplast and chloroplast fractions, purified on Percoll discontinuous gradient, were phosphorylated in vitro using (γ-³²P)ATP, resolved by SDS-PAGE and autoradiographed. In general, about 15-18 phosphopolypeptides in the range of 14-150 kD were distinctly visible in autoradiograms of both organelle fractions with varying degree of radiolabel incorporation. Although short-term irradiation with red or far-red light did not have any significant effect on phosphorylation status of etioplast polypeptides, in vivo irradiation with 1 h white light, followed by in vitro phosphorylation, decreased phosphorylation of a 116 kD polypeptide and increased the phosphorylation of polypeptides of 38 kD and a doublet around 20 kD. Strikingly, the phosphorylation status of 116 kD etioplast polypeptide was adversely affected by Ca²⁺ as well, and this phosphopolypeptlde was not distinctly visible in the autoradiogram of the chloroplast fraction proteins. However, in vitro phosphorylation of 98, 57 and 50 kD polypeptides of both etioplast and chloroplast fractions was found to be Ca²⁺ dependent. Unlike Ca²⁺, 3′,5′-cyclic AMP down-regulated the phosphorylation of several polypeptides of both etioplasts and chloroplasts, including 98 and 50 kD, and up-regulated the phosphorylation of 32 and 57 kD polypeptides. The significance of these observations on changes in phosphoprotein profile of etioplasts and chloroplasts, as influenced by light, Ca²⁺ and cyclic nucleotides, has been discussed.     

Identification of proteins regulated by ABA in response to combined drought and heat stress in maize roots

We adopted a proteomics approach to identify and analyze the differential expression of maize root proteins associated with abscisic acid (ABA) regulation under combined drought and heat stress. Using mass spectrometry, we identified 22 major proteins that were significantly up-regulated under combined drought and heat stress. These 22 proteins were classified into 6 functional categories: disease/defense (8), metabolism (3), cell growth/division (3), signal transduction (2), transporters (2) and unclassified (4). Our previous reports showed that ABA regulates the expression of several small heat-shock proteins (sHSPs) in maize leaves subjected to the combination of drought and heat stress; however, no sHSPs were identified among the root proteins up-regulated in this study. RT-PCR and western blot analyses were used to identify six known sHSPs. The maize roots were pretreated with 100 μM of ABA, and subsequently, the expression of the 22 up-regulated proteins and 6 sHSPs was examined. 11 proteins were up-regulated in an ABA-dependent manner, 13 proteins were up-regulated in an ABA-independent manner, and 4 proteins were up-regulated but inhibited by ABA. The up-regulated proteins are interesting candidates for further physiological and molecular investigations of combination stress tolerance in maize.     

Heat shock protein 70 regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress

We investigated the interaction between heat shock protein 70 (HSP70) and abscisic acid (ABA)-induced antioxidant response of maize to the combination of drought and heat stress. First, the increased activities of enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), induced by drought were less than those by heat or combined drought and heat stress, except some individual cases (e.g. CAT in leaves, GR in roots). Second, both HSP70 synthesis and H₂O₂ production increased prominently under drought, heat or their combination stress; the increase in leaves induced by drought and heat combination was the highest, followed by heat and by drought, while the increase in roots had not visible difference. Third, either in leaves or roots, pretreatment with ABA inhibitor, HSP70 inhibitor and H₂O₂ scavenger, significantly arrested the stress-induced increase of antioxidant enzyme activities, and ABA inhibitor and H₂O₂ scavenger obviously suppressed HSP70 synthesis, while HSP70 inhibitor slightly heightened H₂O₂ accumulation. Finally, 100 μM ABA significantly enhanced the activities of antioxidant enzymes, HSP70 expression and H₂O₂ production under stresses in comparison with ABA-deficient mutant vp5 maize plants without pretreatment. Thus, ABA-induced H₂O₂ production enhances the HSP70 synthesis and up-regulates the activities of antioxidant enzymes, resulting in the suppression of cellular reactive oxygen species (ROS) levels. Our results suggest that HSP70 may play a crucial role in ABA-induced antioxidant defense of maize to drought and heat combination.     

Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 β, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

The maize viviparous15 locus encodes the molybdopterin synthase small subunit

A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15-specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5' untranslated region as well as a micro-synteny among the cereals.