Regulators of early maize leaf development inferred from transcriptomes of laser capture microdissection (LCM)-isolated embryonic leaf cells.

The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.

Maize Golden2-like transcription factors boost rice chloroplast development, photosynthesis, and grain yield.

Chloroplasts are the sites for photosynthesis, and two Golden2-like factors act as transcriptional activators of chloroplast development in rice (Oryza sativa L.) and maize (Zea mays L.). Rice OsGLK1 and OsGLK2 are orthologous to maize ZmGLK1 (ZmG1) and ZmGLK2 (ZmG2), respectively. However, while rice OsGLK1 and OsGLK2 act redundantly to regulate chloroplast development in mesophyll cells, maize ZmG1 and ZmG2 are functionally specialized and expressed in different cell-specific manners. To boost rice chloroplast development and photosynthesis, we generated transgenic rice plants overexpressing ZmG1 and ZmG2, individually or simultaneously, with constitutive promoters (pZmUbi::ZmG1 and p35S::ZmG2) or maize promoters (pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2). Both ZmG1 and ZmG2 genes were highly expressed in transgenic rice leaves. Moreover, ZmG1 and ZmG2 showed coordinated expression in pZmG1::ZmG1/pZmG2::ZmG2 plants. All Golden2-like (GLK) transgenic plants had higher chlorophyll and protein contents, Rubisco activities and photosynthetic rates per unit leaf area in flag leaves. However, the highest grain yields occurred when maize promoters were used; pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1/pZmG2::ZmG2 transgenic plants showed increases in grain yield by 51%, 47%, and 70%, respectively. In contrast, the pZmUbi::ZmG1 plant produced smaller seeds without yield increases. Transcriptome analysis indicated that maize GLKs act as master regulators promoting the expression of both photosynthesis-related and stress-responsive regulatory genes in both rice shoot and root. Thus, by promoting these important functions under the control of their own promoters, maize GLK1 and GLK2 genes together dramatically improved rice photosynthetic performance and productivity. A similar approach can potentially improve the productivity of many other crops.

Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth.

Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.

Whole-Genome Duplication Facilitated the Evolution of C4 Photosynthesis in Gynandropsis gynandra.

In higher plants, whole-genome duplication (WGD) is thought to facilitate the evolution of C4 photosynthesis from C3 photosynthesis. To understand this issue, we used new and existing leaf-development transcriptomes to construct two coding sequence databases for C4Gynandropsis gynandra and C3Tarenaya hassleriana, which shared a WGD before their divergence. We compared duplicated genes in the two species and found that the WGD contributed to four aspects of the evolution of C4 photosynthesis in G. gynandra. First, G. gynandra has retained the duplicates of ALAAT (alanine aminotransferase) and GOGAT (glutamine oxoglutarate aminotransferase) for nitrogen recycling to establish a photorespiratory CO2 pump in bundle sheath (BS) cells for increasing photosynthesis efficiency, suggesting that G. gynandra experienced a C3-C4 intermediate stage during the C4 evolution. Second, G. gynandra has retained almost all known vein-development-related paralogous genes derived from the WGD event, likely contributing to the high vein complexity of G. gynandra. Third, the WGD facilitated the evolution of C4 enzyme genes and their recruitment into the C4 pathway. Fourth, several genes encoding photosystem I proteins were derived from the WGD and are upregulated in G. gynandra, likely enabling the NADH dehydrogenase-like complex to produce extra ATPs for the C4 CO2 concentration mechanism. Thus, the WGD apparently played an enabler role in the evolution of C4 photosynthesis in G. gynandra. Importantly, an ALAAT duplicate became highly expressed in BS cells in G. gynandra, facilitating nitrogen recycling and transition to the C4 cycle. This study revealed how WDG may facilitate C4 photosynthesis evolution.

Comparative transcriptomics method to infer gene coexpression networks and its applications to maize and rice leaf transcriptomes.

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Chromosomal-level genome assembly of the semi-dwarf rice Taichung Native 1, an initiator of Green Revolution.

Here we report the 409.5 Mb chromosome-level assembly of the first bred semi-dwarf rice, the Taichung Native 1 (TN1), which served as the template for the development of the Green Revolution (GR) cultivar IR8 "miracle rice". We sequenced the TN1 genome utilizing multiple platforms and produced PacBio long reads, Illumina paired-end reads, Illumina mate-pair reads and 10x Genomics linked reads. We used a hybrid approach to assemble the 226× coverage of sequences by a combination of de novo and reference-guided approaches. The assembled TN1 genome has an N50 scaffold size of 33.1 Mb with the longest measuring 45.5 Mb. We annotated 37,526 genes, in which 24,102 (64.23%) were assigned Blast2GO annotations. The genome has 4672 or 95.4% complete BUSCOs and a repeat content of 51.52%. We developed our own method of creating a GR pangenome using the orthologous relationships of the proteins of TN1, IR8, MH63 and IR64, identifying 16,999 core orthologue groups of Green Revolution. From the pangenome, we identified a set of shared and unique gene ontology terms for the accessory clusters, characterizing TN1, IR8, MH63 and IR64. This TN1 genome assembly and GR pangenome will be a resource for new genomic discoveries about Green Revolution, and for improving the disease and insect resistances and the yield of rice.

Elevated auxin biosynthesis and transport underlie high vein density in C4 leaves.

High vein density, a distinctive trait of C4 leaves, is central to both C3-to-C4 evolution and conversion of C3 to C4-like crops. We tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. Up-regulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C4 leaves compared with developing C3 leaves. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Moreover, auxin content and vein density were increased in loss-of-function mutants of Arabidopsis MYC2, a suppressor of auxin biosynthesis. Treatment with an auxin biosynthesis inhibitor or an auxin transport inhibitor led to much fewer veins in new leaves. Finally, both Arabidopsis thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. Thus, development of high leaf vein density requires elevated auxin biosynthesis and transport.

Hydrogen cyanamide breaks grapevine bud dormancy in the summer through transient activation of gene expression and accumulation of reactive oxygen and nitrogen species.

BACKGROUND: Hydrogen cyanamide (HC) and pruning (P) have frequently been used to break dormancy in grapevine floral buds. However, the exact underlying mechanism remains elusive. This study aimed to address the early mode of action of these treatments on accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and expression of related genes in the dormancy breaking buds of grapevine in the summer. RESULTS: The budbreak rates induced by pruning (P), hydrogen cyanamide (HC), pruning plus hydrogen cyanamide (PHC) and water (control) after 8 days were 33, 53, 95, and 0 %, respectively. Clearly, HC was more effective in stimulating grapevine budbreak and P further enhanced its potency. In situ staining of longitudinal bud sections after 12 h of treatments detected high levels of ROS and nitric oxide (NO) accumulated in the buds treated with PHC, compared with HC or P alone. The amounts of ROS and NO accumulated were highly correlated with the rates of budbreak among these treatments, highlighting the importance of a rapid, transient accumulation of sublethal levels of ROS and RNS in dormancy breaking. Microarray analysis revealed specific alterations in gene expression in dormancy breaking buds induced by P, HC and PHC after 24 h of treatment. Relative to control, PHC altered the expression of the largest number of genes, while P affected the expression of the least number of genes. PHC also exerted a greater intensity in transcriptional activation of these genes. Gene ontology (GO) analysis suggests that alteration in expression of ROS related genes is the major factor responsible for budbreak. qRT-PCR analysis revealed the transient expression dynamics of 12 specific genes related to ROS generation and scavenge during the 48 h treatment with PHC. CONCLUSION: Our results suggest that rapid accumulation of ROS and NO at early stage is important for dormancy release in grapevine in the summer, and the identification of the commonly expressed specific genes among the treatments allowed the construction of the signal transduction pathway related to ROS/RNS metabolism during dormancy release. The rapid accumulation of a sublethal level of ROS/RNS subsequently induces cell wall loosening and expansion for bud sprouting.

A variation on chloroplast development: the bizonoplast and photosynthetic efficiency in the deep-shade plant Selaginella erythropus.

UNLABELLED: • PREMISE OF THE STUDY: Chloroplast development and structure are highly conserved in vascular plants, but the bizonoplast of Selaginella is a notable exception. In the shade plant S. erythropus, each dorsal epidermal cell contains one bizonoplast, while other cells have normal chloroplasts. Our quest was to (1) determine the origin of bizonoplasts, (2) explore developmental plasticity, and (3) correlate developmental changes with photosynthetic activity to provide insights unavailable in other green plants with more constrained development.• METHODS: Bizonoplast development was studied in juvenile prostrate and older erect shoots of S. erythropus. Plastid plasticity was studied in plants cultivated under different light conditions. Chlorophyll fluorescence was measured and correlated with photosynthetic activity.• KEY RESULTS: The bizonoplast originates from a proplastid, forming a distinctive upper zone rapidly after exposure to low light. In the prostrate shoots, the proplastid develops through early stages only. When the shoot becomes erect, the proplastid soon develops into a mature bizonoplast. Erect shoots have significantly higher photosynthetic efficiency than prostrate shoots. No bizonoplasts were found in the plants growing in high light, where 2-4 spheroidal chloroplasts formed, or with light from below.• CONCLUSIONS: The upper zone develops above a normal-looking chloroplast structure to produce a bizonoplast. Bizonoplast developmental plasticity suggests that regular lamellar structure and monoplastidy are adaptations to deep shade environments. Such novel variation in S. erythropus is in stark contrast to known plastid development in other vascular plants, possibly reflecting retention of developmental flexibility in the basal clade, Lycophyta, to which it belongs.

Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis.

To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C(4) metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.

C4 leaf development and evolution.

C4 photosynthesis is more efficient than C3 photosynthesis for two reasons. First, C4 plants have evolved efficient C4 enzymes to suppress wasteful photorespiration and enhance CO2 fixation. Second, C4 leaves have Kranz anatomy in which the veins are surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. The BS and M cells are functionally well differentiated and also well coordinated for rapid assimilation of atmospheric CO2 and transport of photo-assimilates between the two types of cells. Recent comparative transcriptomics of developing M and BS cells in young maize embryonic leaves revealed not only potential regulators of BS and M cell differentiation but also rapid early BS cell differentiation whereas slower, more prolonged M cell differentiation, contrary to the traditional view of a far simpler process of M cell development. Moreover, new upstream regulators of Kranz anatomy development have been identified and a number of gene co-expression modules for early vascular development have been inferred. Also, a candidate gene regulatory network associated with Kranz anatomy and vascular development has been constructed. Additionally, how whole genome duplication (WGD) may facilitate C4 evolution has been studied and the reasons for why the same WGD event led to successful C4 evolution in Gynandropsis gynandra but not in the sister species Tarenaya hassleriana have been proposed. Finally, new future research directions are suggested.

Characterization of a salt-induced DhAHP, a gene coding for alkyl hydroperoxide reductase, from the extremely halophilic yeast Debaryomyces hansenii.

BACKGROUND: Debaryomyces hansenii is one of the most salt tolerant species of yeast and has become a model organism for the study of tolerance mechanisms against salinity. The goal of this study was to identify key upregulated genes that are involved in its adaptation to high salinity. RESULTS: By using forward subtractive hybridization we have cloned and sequenced DhAHP from D. hansenii that is significantly upregulated during salinity stress. DhAHP is orthologous to the alkly hydroperoxide reductase of the peroxiredoxin gene family, which catalyzes the reduction of peroxides at the expense of thiol compounds. The full-lengthed cDNA of DhAHP has 674 bp of nucleotide and contains a 516 bp open reading frame (ORF) encoding a deduced protein of 172 amino acid residues (18.3 kDa). D. hansenii Ahp is a cytosolic protein that belongs to the Ahp of the 1-Cys type peroxiredoxins. Phylogentically, the DhAhp and Candida albicans Ahp11 (Swiss-Prot: Q5AF44) share a common ancestry but show divergent evolution. Silence of its expression in D. hansenii by RNAi resulted in decreased tolerance to salt whereas overexpression of DhAHP in D. hansenii and the salt-sensitive yeasts Saccharomyces cereviasiae and Pichia methanolica conferred a higher tolerance with a reduced level of reactive oxygen species. CONCLUSION: In conclusion, for the first time our study has identified alkly hydroperoxide reductase as a key protein involved in the salt tolerance of the extremely halophilic D. hansenii. Apparently, this enzyme plays a multi-functional role in the yeast's adaptation to salinity; it serves as a peroxidase in scavenging reactive oxygen species, as a molecular chaperone in protecting essential proteins from denaturation, and as a redox sensor in regulating H2O2-mediated cell defense signaling.

Assembling the Setaria italica L. Beauv. genome into nine chromosomes and insights into regions affecting growth and drought tolerance.

The diploid C4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains.

Down-Regulation of Cytokinin Oxidase 2 Expression Increases Tiller Number and Improves Rice Yield.

BACKGROUND: Cytokinins are plant-specific hormones that affect plant growth and development. The endogenous level of cytokinins in plant cells is regulated in part by irreversible degradation via cytokinin oxidase/dehydrogenase (CKX). Among the 11 rice CKXs, CKX2 has been implicated in regulation of rice grain yield. RESULTS: To specifically down-regulate OsCKX2 expression, we have chosen two conserved glycosylation regions of OsCKX2 for designing artificial short hairpin RNA interference genes (shRNA-CX3 and -CX5, representing the 5' and 3' glycosylation region sequences, respectively) for transformation by the Agrobacterium-mediated method. For each construct, 5 independent transgenic lines were obtained for detailed analysis. Southern blot analysis confirmed the integration of the shRNA genes into the rice genome, and quantitative real time RT-PCR and northern blot analyses showed reduced OsCKX2 expression in the young stem of transgenic rice at varying degrees. However, the expression of other rice CKX genes, such as CKX1 and CKX3, in these transgenic lines was not altered. Transgenic rice plants grown in the greenhouse were greener and more vigorous with delayed senescence, compared to the wild type. In field experiments, both CX3 and CX5 transgenic rice plants produced more tillers (27-81 %) and grains (24-67 %) per plant and had a heavier 1000 grain weight (5-15 %) than the wild type. The increases in grain yield were highly correlated with increased tiller numbers. Consistently, insertional activation of OsCKX2 led to increased expression of CKX2 and reduced tiller number and growth in a gene-dosage dependant manner. CONCLUSIONS: Taken together, these results demonstrate that specific suppression of OsCKX2 expression through shRNA-mediated gene silencing leads to enhanced growth and productivity in rice by increasing tiller number and grain weight.

Oxygen sensitivity of photosynthesis and photorespiration in different photosynthetic types in the genus Flaveria.

Two major indicators were used to access the degree of photorespiration in various photosynthetic types of Flaveria species (C3, C3-C4, C4-like, and C4): the O2 inhibition of photosynthesis measured above the O2 partial pressure which gives a maximum rate, and O2- and light-dependent whole-chain electron flow measured at the CO2 compensation point (Γ). The optimum level of O2 for maximum photosynthetic rates under atmospheric levels of CO2 (34 Pa) was lower in C3 and C3-C4 species (ca. 2 kPa) than in C4-like and C4 species (ca. 9 kPa). Increasing O2 partial pressures from the optimum for photosynthesis up to normal atmospheric levels (ca. 20 kPa) caused an inhibition of photosynthesis which was more severe under lower CO2. This inhibition was calculated as the O2 inhibition index (ΘA, the percentage inhibition of photosynthesis per kPa increase in O2). From measurements of 18 Flaveria species at atmospheric CO2, the ΘA values decreased from C3 (1.9-2.1) to C3-C4 (1.2-1.6), C4-like (0.6-0.8) and C4 species (0.3-0.4), indicating a progressive decrease in apparent photorespiration in this series. With increasing irradiance at Γ under atmospheric levels of O2, and increasing O2 partial pressure at 300 µmol quanta·m-2·s-1, there was a similar increase in the rate of O2 evolution associated with whole-chain electron flow (Jo2, calculated from chlorophyll fluorescence analysis) in the C3 and C3-C4 species compared to a much lower rate in the C4-like and C4 species. The results indicate that there is substantial O2-dependent electron flow in C3 and C3-C4 species, reflecting a high level of photorespiration compared to that in C4-like and C4 species. Consistent with these results, there was a significant decrease in Γ from C3 (6-6.2 Pa) to C3-C4 (1.0-3.0 Pa), to C4-like and C4 species (0.3-0.8 Pa), indicating a progressive decrease in apparent photorespiration. However, C3 and C3-C4 species examined had high intrinsic levels of photorespiration with the latter maintaining low apparent rates of photorespiration and lower Γ values, primarily by refixing photorespired CO2. The C4-like and C4 Flaveria species had low, but measurable, levels of photorespiration via selective localization of ribulose-1,5-bisphosphate carboxylase in bundle sheath cells and operation of a CO2 pump via the C4 pathway.

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C(4) photosynthesis enzymes.

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO(2) uptake rate and carboxylation efficiency, and slightly reduced CO(2) compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22-24% more grains than WT plants. Taken together, these results suggest that expression of maize C(4) photosynthesis enzymes in rice, a C(3) plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation.

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds.

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.

Enhancement of temozolomide-induced apoptosis by valproic acid in human glioma cell lines through redox regulation.

Temozolomide (TMZ) is an oral alkylating agent that has been widely used in the treatment of refractory glioma, although inherent and acquired resistance to this drug is common. The clinical use of valproic acid (VPA) as an anticonvulsant and mood-stabilizing drug has been reported primarily for the treatment of epilepsy and bipolar disorder and less commonly for major depression. VPA is also used in the treatment of glioma-associated seizures with or without intracranial operation. In this study, we evaluated the potential synergistic effect of TMZ and VPA in human glioma cell lines. Compared with the use of TMZ or VPA alone, concurrent treatment with both drugs synergistically induced apoptosis in U87MG cells as evidenced by p53 and Bax expression, mitochondrial transmembrane potential loss, reactive oxygen species production, and glutathione depletion. This synergistic effect correlated with a decrease in nuclear translocation of the nuclear factor-erythroid 2 p45-related factor and corresponded with reduced heme oxygenase-1 and γ-glutamylcysteine synthetase expression. Pretreatment with N-acetylcysteine partially recovered the apoptotic effect of the TMZ/VPA combination treatment. The same degree of synergism is also seen in p53-mutant Hs683 cells, which indicates that p53 may not play a major role in the increased proapoptotic effect of the TMZ/VPA combination. In conclusion, VPA enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism. The TMZ/VPA combination may be effective for treating glioma cancer and may be a powerful agent against malignant glioma. This drug combination should be further explored in the clinical setting.