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Background: NAD kinases (NADKs) are the only known enzymes that directly phosphorylate NAD(H) to generate NADP(H) in different subcellular compartments. They participate in multiple life activities, such as modulating the NADP/NAD ratio, maintaining the intracellular redox balance and responding to environmental stresses. However, the functions of individual NADK in plants are still under investigation. Here, a rice NADK, namely, OsNADK1, was identified, and its functions in plant growth regulation and stress tolerance were analysed by employing a series of transgenic plant lines., Results: OsNADK1 is a cytosol-localized NADK in rice. It was expressed in all rice tissues examined, and its transcriptional expression could be stimulated by a number of environmental stress treatments. Compared with wild-type (WT) rice, the mutant plant osnadk1 in which OsNADK1 was knocked out was a dwarf at the heading stage and had decreased NADP(H)/NAD(H), ascorbic acid (ASA)/dehydroascorbate (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios, which led to increased oxidation states in the rice cells and sensitivity to drought. Moreover, certain stress-related genes showed differential expression patterns in osnadk1 under both normal growth and drought-stress conditions compared with WT. Among these genes, OsDREB1B and several WRKY family transcription factors, e.g., OsWRKY21 and OsWRKY42, showed correlated co-expression patterns with OsNADK1 in osnadk1 and the plants overexpressing or underexpressing OsNADK1, implying roles for these transcription factors in OsNADK1-mediated processes. In addition, overexpression of OsNADK1 enhanced the drought tolerance of rice plants, whereas loss of function of the gene reduced the tolerance. Furthermore, the proline content was dramatically increased in the leaves of the OsNADK1-overexpressing lines under drought conditions., Conclusions: Altogether, the results suggest that an OsNADK1-mediated intracellular redox balance is involved in the tolerance of rice plants to drought.

We describe a protocol for transient gene expression in rice protoplasts and its application to the study of Magnaporthe oryzae avirulence (AVR) gene function. In this assay the gene encoding the firefly luciferase protein is transfected into rice protoplasts by electroporation together with the candidate AVR genes. The luminescence can then be used to assess the viability of rice protoplasts. The hypersensitive response (HR) caused by the interaction between M. oryzae AVR and rice R genes can subsequently be monitored by recording the decrease in luminescence from the transfected cells.

Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100 x g 2 min and the protoplasts yield was 7x106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100 x g 2 min and the protoplasts yield was 6 x 10(6) cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000 x g 2 min and the protoplasts yield was 6x10(6) cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%-80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.

Here, we describe a series of methods suitable for the measurement of cytosolic ion concentrations in living plant cells using ion selective dyes. We describe procedures for the use of SBFI for the measurement of Na(+) in live cells. The resulting material is suitable for most standard cell biology procedures.

Ca(2+) and calmodulin (CaM) have been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense. However, it is unknown whether Ca(2+)/CaM-dependent protein kinase (CCaMK) is involved in the process. In the present study, the role of rice CCaMK, OsDMI3, in ABA-induced antioxidant defense was investigated in leaves of rice (Oryza sativa) plants. Treatments with ABA, H(2)O(2), and polyethylene glycol (PEG) induced the expression of OsDMI3 and the activity of OsDMI3, and H(2)O(2) is required for the ABA-induced increases in the expression and the activity of OsDMI3 under water stress. Subcellular localization analysis showed that OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane. The analysis of the transient expression of OsDMI3 in rice protoplasts and the RNA interference (RNAi) silencing of OsDMI3 in rice protoplasts showed that OsDMI3 is required for ABA-induced increases in the expression and the activities of superoxide dismutase (SOD) and catalase (CAT). Further, the oxidative damage induced by higher concentrations of PEG and H(2)O(2) was aggravated in the mutant of OsDMI3. Moreover, the analysis of the RNAi silencing of OsDMI3 in protoplasts and the mutant of OsDMI3 showed that higher levels of H(2)O(2) accumulation require OsDMI3 activation in ABA signaling, but the initial H(2)O(2) production induced by ABA is not dependent on the activation of OsDMI3 in leaves of rice plants. Our data reveal that OsDMI3 is an important component in ABA-induced antioxidant defense in rice.

3' untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. We evaluated the potential of the 3'-UTRs of 9 seed storage protein (SSP) genes as terminators in enhancing the expression of the β-glucuronidase (gus A) reporter gene driven by the glutelin GluB-3 promoter in stable transgenic rice lines. Six of the 3'-UTRs significantly enhanced the activity of the GluB-3 promoter without changing its tissue specificity but altered its expression pattern in endosperm. With the 3'-UTRs of GluB-5, GluA-2 and GluC, the expression of gus A was higher by 3.12-, 2.45- and 2.14-fold, respectively, than with the Nos terminator. These three 3'-UTRs, combined with GluC, Ubi-1 and CaMV35S promoters, also increased GUS levels in stable transgenic rice lines or in transient expression in protoplasts, which indicated that the enhancements were independent of the promoter sequence. The increase in protein production was accompanied by altered mRNA levels, which suggests that the enhancements were due to increased transcript level. The 3'-UTRs of GluB-5, GluA-2 and GluC, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds. The 9 SSP 3'-UTRs could function as faithful terminators in mono- or multi-gene transformation avoiding homology-based gene silencing.

Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid β-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased α-carotene, while Chl a/b levels were unaltered. The increased α-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in α-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased α-carotene, contributing to sensitivity to intense light.

Salinity poses a major threat for agriculture worldwide. Rice is one of the major crops where most of the high-yielding cultivars are highly sensitive to salinity. Several studies on the genetic variability across rice cultivars suggest that the activity and composition of root plasma membrane transporters could underlie the observed cultivar-specific salinity tolerance in rice. In the current study, it was found that the salt-tolerant cultivar Pokkali maintains a higher K+/Na+ ratio compared with the salt-sensitive IR20 in roots as well as in shoots. Using Na+ reporter dyes, IR20 root protoplasts showed a much faster Na+ accumulation than Pokkali protoplasts. Membrane potential measurements showed that root cells exposed to Na+ in IR20 depolarized considerably further than those of Pokkali. These results suggest that IR20 has a larger plasma membrane Na+ conductance. To assess whether this could be due to different ion channel properties, root protoplasts from both Pokkali and IR20 rice cultivars were patch-clamped. Voltage-dependent K+ inward rectifiers, K+ outward rectifiers, and voltage-independent, non-selective channels with unitary conductances of around 35, 40, and 10 pS, respectively, were identified. Only the non-selective channel showed significant Na+ permeability. Intriguingly, in both cultivars, the activity of the K+ inward rectifier was drastically down-regulated after plant growth in salt but gating, conductance, and activity of all channel types were very similar for the two cultivars.

Tapetum development and meiosis play crucial roles in anther development. Here we identified a rice gene, DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), which controls the early stages of that development. This gene encodes for an endoplasmic reticulum (ER) membrane protein that is present only in cereals. Our T-DNA insertion mutations gave rise to abnormal tapetal formation. Cellular organelles, especially the ER, were underdeveloped, which led to hampered differentiation and degeneration of the tapetum. In addition, the development of pollen mother cells was arrested at the early stages of meiotic prophase I. RNA in-situ hybridization analyses showed that DTM1 transcripts were most abundant in tapetal cells at stages 6 and 7, and moderately in the pollen mother cells and meiocytes. Transcripts of UDT1, which functions in tapetum development during early meiosis, were reduced in dtm1 anthers, as were those of PAIR1, which is involved in chromosome pairing and synapsis during meiosis. However, expression of MSP1 and MEL1, which function in anther wall specification and germ cell division, respectively, was not altered in the dtm1 mutant. Moreover, transcripts of DTM1 were reduced in msp1 mutant anthers, but not in udt1 and pair1 mutants. These results, together with their mutant phenotypes, suggest that DTM1 plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Imazethapyr (IM) is a chiral herbicide and a widely used racemic mixture. This report investigated the enantioselectivity between R- and S-IM in rice and explored its causative mechanism at the physiological and molecular levels. The results suggested that R-IM inhibited acetolactate synthase (ALS) activity to a greater extent than S-IM, which reduced the synthesis of branched-chain amino acids (BCAAs). Additionally, most other amino acids showed enantioselectivity. On the cellular level, R-IM showed stronger toxicity against protoplasts than S-IM. A gene transcription profile analysis showed that gene transcripts in many metabolic pathways, including amino acid metabolism, photosynthesis, starch and sugar metabolism, and the tricarboxylic acid cycle displayed enantioselectivity between the IM enantiomers. R-IM regulated more genes more strongly than S-IM. This study suggested that R-IM has stronger toxicity against plants than S-IM; this toxicity is caused not only by changing targeted enzyme activity and amino acid synthesis, but also by affecting gene transcription in other metabolic pathways directly or indirectly in an enantioselective manner.

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H(2)O(2)-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ(m)) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ(m) relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ(m) and inhibiting the amplification of ROS., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. To date, the techniques that are available to measure cGMP in plants are limited by low spatial and temporal resolution. In addition, tissue destruction is necessary. To circumvent these drawbacks we have used the δ-FlincG fluorescent protein to create an endogenous cGMP sensor that can report cellular cGMP levels with high resolution in time and space in living plant cells. δ-FlincG in transient and stably expressing cells shows a dissociation constant for cGMP of around 200 nm giving it a dynamic range of around 20-2000 nm. Stimuli that were previously shown to alter cGMP in plant cells (nitric oxide and gibberrellic acid) evoked pronounced fluorescence signals in single cells and in root tissues, providing evidence that δ-FlincG reports changes in cellular cGMP in a physiologically relevant context., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Although there is evident homology among reproductive organs when comparing Poaceae (grass) and eudicots, the identity of grass specific organs, such as lodicules, palea, lemma, and glumes has been the subject of a vast and largely inconclusive discussion. Here we provide some direct evidence to support the idea that the empty glumes of rice (Oryza sativa) are counterparts of lemmas. We show that the development of empty glumes is regulated by ELE (elongated empty glume), which belongs to a plant specific novel gene family. Mutations at the ELE locus cause elongated empty glumes, which mimic the lemmas and have the epidermal morphology of lemmas with four or five vascular bundles. As a nuclear-localized gene, ELE is specifically expressed at the empty glumes of immature spikelets, and its ectopic expression causes many floral development defects, including lemma-like palea, extra palea-like structures, elongated lodicules, extra stamens and stigmas. Our result suggests that empty glumes are lemmas of the sterile florets located at the lateral side of the rice spikelet, and ELE acts as a regulator restraining its growth to maintain its small size in wild-type plants., (Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Because protein synthesis begins with N-formylmethionine in plant endosymbiotic organelles, removal of the formyl group by peptide deformylase (PDF) is essential to allowing the excision of the first methionine. Rice contains three copies (OsPDF1A, OsPDF1B and OsPDF1B2) of the PDF genes. Unlike OsPDF1A and OsPDF1B, OsPDF1B2 is apparently non-functional, with several deleterious substitutions and deletions. OsPDF1A is more strongly expressed in the roots, while OsPDF1B is expressed at higher levels in mature leaves. Transient expression of PDF-green fluorescent protein (GFP) fusion proteins in the protoplasts demonstrates that, unlike OsPDF1A, OsPDF1B is localized in both the chloroplasts and the mitochondria. We used T-DNA insertional alleles to elucidate functional roles associated with OsPDF1B. Homozygous plants of pdf1b/pdf1b exhibited the phenotypes of chlorina and growth retardation. Histochemical analysis showed that the length of their mesophyll cells was increased 4- to 5-fold, resulting in a reduction in the total number of cells. Transmission electron microscopy analyses revealed that chloroplasts were severely damaged and mitochondria appeared to be mildly altered in the pdf1b mutants. Expression of genes encoded in the chloroplasts and mitochondria was altered in the mutants. Based on these results, we conclude that OsPDF1B is essential for the development of chloroplast and perhaps mitochondria.

The eukaryotic chromatin/chromosome stores genomic information, controls genetic material distribution, and plays an essential role in the establishment and maintenance of spatial and temporal gene expression profile. Despite over a century of research, the protein composition and higher level structure of chromatin still remain obscure, particularly in plants. In this report, we have developed a protocol for chromatin purification from rice suspension cells and examined proteins copurified with chromatin using both 2-DE gel and shotgun approaches. Nine hundred seventy-two distinct protein spots have been resolved on 2-DE gels and 509 proteins have been identified by MALDI-MS/MS following gel excision, which correspond to 269 unique proteins. When the chromatin copurified proteins are examined using shotgun method, a large number of histone variants in addition to the four common core histones have been identified. Other proteins identified include nucleosome assembly proteins, high mobility group proteins, histone modification proteins, transcription factors, and a large number of hypothetical and function unknown proteins. Furthermore, putative phosphoproteins copurified with chromatin have been examined using Pro-Q Diamond phosphoprotein stain and followed by MALDI-MS/MS. Our studies have provided valued new insight into chromatin composition in plants.

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.

In this paper, we report a novel pentatricopeptide repeat (PPR) protein gene in rice. PPR, a characteristic repeat motif consisted of tandem 35 amino acids, has been found in various biological systems including plant. Sequence analysis revealed that the gene designated OsPPR1 consisted of an open reading frame of 2433 nucleotides encoding 810 amino acids that include 11 PPR motifs. Blast search result indicated that the gene did not align with any of the characterized PPR genes in plant. The OsPPR1 gene was found to contain a putative chloroplast transit peptide in the N-terminal region, suggesting that the gene product targets to the chloroplast. Southern blot hybridization indicated that the OsPPR1 is the member of a gene family within the rice genome. Expression analysis and immunoblot analysis suggested that the OsPPR1 was accumulated mainly in rice leaf. Antisense transgenic strategy was used to suppress the expression of OsPPR1 and the resulted transgenic rice showed the typical phenotypes of chlorophyll-deficient mutants; albinism and lethality. Cytological observation using microscopy revealed that the antisense transgenic plant contained a significant defect in the chloroplast development. Taken together, the results suggest that the OsPPR1 is a nuclear gene of rice, encoding the PPR protein that might play a role in the chloroplast biogenesis. This is the first report on the PPR protein required for the chloroplast biogenesis in rice.

The beneficial effects have been studied of supplementing culture medium with 1:100-1:500 (v:v) of a commercial haemoglobin solution (Erythrogen) on the mitotic division of cell suspension-derived protoplasts of Indica rices (Oryza sativa L.). Protoplasts were cultured in liquid medium, at densities of 1.5 x 10(6) or 2.5 x 10(6) ml(-1), on nitrocellulose membranes overlaying a semi-solidified medium layer that was supplemented with both Erythrogen and nurse cells of Lolium multiflorum. The mean final plating efficiencies (FPEs) of rice cv. BR26 protoplasts cultured with 1:200 (v:v) Erythrogen, at 1.5 x 10(6) ml(-1) (0.018+/-0.003%; n = 8) and 2.5 x 10(6) ml(-1) (0.016+/-0.002%; n = 8), were both significantly (P < 0.05) greater than controls lacking Erythrogen (0.0058+/-0.002%; n = 8 and 0.0041+/-0.001%; n = 8, respectively). Similarly, the mean FPEs of cv. Bini protoplasts cultured with 1:200 (v:v) Erythrogen at 1.5 x 10(6) ml(-1) (0.012+/-0.003%; n = 6) and 2.5 x 10(6) ml(-1) (0.017+/-0.001%; n = 6) were also significantly (P < 0.05) greater than their respective controls (0.003+/-0.001%, n = 6 and 0.002+/-0.001%, n = 6). In contrast, supplementation with 1:100 or 1:500 (v:v) Erythrogen did not lead to sustained mitotic division and microcallus formation in both rice cultivars.

Transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of Basmati rice because they enable the introduction of one or more genes into a leading cultivar without affecting its genetic background. During the last ten years, a rapid progress has been made towards the development of transformation methods in rice. Several transformation methods including Agrobacterium, biolistic, and DNA uptake by protoplasts, have been employed to produce transgenic rice. An array of useful genes is now available and many of these have already been transferred in rice to improve the resistance against biotic and abiotic stresses. In Basmati rice, a beginning has already been made regarding the development of tissue culture protocols, transformation methods and production of useful transgenic plants. The application and future prospects of transformation technology to engineer the resistance against insect pests (stem borer, leaf folder, brown plant hopper, gall midge), fungal diseases (blast, bakanae/foot, rot), bacterial diseases (bacterial leaf blight, sheath blight), abiotic stresses (salinity and drought) and improved nutritional quality (accumulation of provitamin A and essential amino acids in endosperm) in Basmati rice, have been addressed.