Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance.

Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana transgenics overexpressing cytosolic CuZn-superoxide dismutase (PaSOD) from poly-extremophile high-altitude Himalayan plant Potentilla atrosanguinea, cytosolic ascorbate peroxidase (RaAPX) from Rheum australe and dual transgenics overexpressing both the genes were developed and analyzed under salt stress. In comparison to wild-type (WT) or single transgenics, the performance of dual transgenics under salt stress was better with higher biomass accumulation and cellulose content. We identified genes involved in cell wall biosynthesis, including nine cellulose synthases (CesA), seven cellulose synthase-like proteins together with other wall-related genes. RNA-seq analysis and qPCR revealed differential regulation of genes (CesA 4, 7 and 8) and transcription factors (MYB46 and 83) involved in secondary cell wall cellulose biosynthesis, amongst which most of the cellulose biosynthesis gene showed upregulation in single (PaSOD line) and dual transgenics at 100 mM salt stress. A positive correlation between cellulose content and H2O2 accumulation was observed in these transgenic lines. Further, cellulose content was 1.6-2 folds significantly higher in PaSOD and dual transgenic lines, 1.4 fold higher in RaAPX lines as compared to WT plants under stress conditions. Additionally, transgenics overexpressing PaSOD and RaAPX also displayed higher amounts of phenolics as compared to WT. The novelty of present study is that H2O2 apart from its role in signalling, it also provides mechanical strength to plants and aid in plant biomass production during salt stress by transcriptional activation of cellulose biosynthesis pathway. This modulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create plants with enhanced cellulose content for biofuel use.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development.

In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.

In vitro flowering associated protein changes in Dendrocalamus hamiltonii.

In Dendrocalamus hamiltonii, conversion of vegetative meristem to a floral meristem was successfully achieved on flower induction medium. A total of 128 differentially expressed proteins were evidenced by 2DE in floral meristem protein profiles. Analysis of 103 proteins through PMF revealed change in abundance in the content of 79 proteins, disappearance and new appearance in the content of 7 and 17 proteins, respectively. MS/MS and subsequent homology search identified 65 proteins that were involved in metabolism (22 proteins), regulatory (11 proteins), signaling and transportation (12 proteins), stress (6 proteins), flowering (8 proteins), and unknown functions (6 proteins). The data suggested that change in metabolism related proteins might be providing nutrient resources for floral initiation in D. hamiltonii. Further, interactive effects of various proteins like bHLH145, B-4c transcription factors (heat stress transcription factor), maturase K, MADS box, zinc finger proteins, and scarecrow-like protein 21 (flowering related), a key enzyme of ethylene biosynthesis SAMS (S-adenosylmethionine synthase) and aminocyclopropane-1-carboxylate synthase, improved calcium signaling related proteins (CML36), and change in phytohormone related proteins such as phosphatase proteins (2c3 and 2c55), which are the positive regulators of gibberellic acid and phytochrome regulation related proteins (DASH, LWD1) might be the possible major regulators of floral transition in this bamboo.

Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress.

Abiotic stresses cause accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) in plants. Sophisticated mechanisms are required to maintain optimum level of H2O2 that acts as signalling molecule regulating adaptive response to salt stress. CuZn-superoxide dismutase (CuZn-SOD) and ascorbate peroxidase (APX) constitute first line of defence against oxidative stress. In the present study, PaSOD and RaAPX genes from Potentilla atrosanguinea and Rheum australe, respectively were overexpressed individually as well as in combination in Arabidopsis thaliana. Interestingly, PaSOD and dual transgenic lines exhibit enhanced lignin deposition in their vascular bundles with altered S:G ratio under salt stress. RNA-seq analysis revealed that expression of PaSOD gene in single and dual transgenics positively regulates expression of lignin biosynthesis genes and transcription factors (NACs, MYBs, C3Hs and WRKY), leading to enhanced and ectopic deposition of lignin in vascular tissues with larger xylem fibres and alters S:G ratio, as well. In addition, transgenic plants exhibit growth promotion, higher biomass production and increased yield under salt stress as compared to wild type plants. Our results suggest that in dual transgenics, ROS generated during salt stress gets converted into H2O2 by SOD and its optimum level was maintained by APX. This basal level of H2O2 acts as messenger for transcriptional activation of lignin biosynthesis in vascular tissue, which provides mechanical strength to plants. These findings reveal an important role of PaSOD and RaAPX in enhancing salt tolerance of transgenic Arabidopsis via increased accumulation of compatible solutes and by regulating lignin biosynthesis.

Embryo sac cellularization defects lead to supernumerary egg cells and twin embryos in Arabidopsis thaliana.

Arabidopsis lines with loss-of-function mutation in Embryo sac-specific Pectin MethylEsterase Inhibitor (Atepmei) gene showed seed sterility with embryo sac cellularization defects. Examination of tissue-cleared mature ovules revealed irregularly positioned nuclei/embryos within the embryo sacs. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of ruthenium red and tannic acid-ferric chloride staining of developing Atepmei mutant ovules showed that cell wall formation and maintenance were altered around embryo sac nuclei, which also coincided with change in the gamete specification. This report implicates the role of cell walls in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

Plant proteomics in India and Nepal: current status and challenges ahead.

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing Potentilla superoxide dismutase.

Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copper-zinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less "mean time to germination" under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Cloning and functional characterization of ß-1, 3-glucanase gene from Podophyllum hexandrum - a high altitude Himalayan plant.

Podophyllum hexandrum is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. ß-1, 3-glucanase cDNA was cloned from the germinating seeds of Podophyllum (Ph-glucanase). Glucanases belong to pathogenesis related glycohydralase family of proteins, which also play an important role in endosperm weakening and testa rupture during seed germination. Analysis of cloned nucleotide sequence revealed Ph-glucanase with an open reading frame of 852bp encoding a protein of 283 amino acids with a molecular mass of 31kDa and pI of 4.39. In-silico structure prediction of Ph-glucanase showed homology with that of Hevea brasiliensis (3em5B). Structural stability and enhanced catalytic efficiency in harsh climatic conditions possibly due to the presence of glycosyl hydrolase motif (LGIVISESGWPSAG) and a connecting loop towards inner side and well exposed carbohydrate metabolism domain-COG5309, can readily hydrolyse cell wall sugar moieties. Seeds from the transgenic Arabidopsis plants over-expressing Ph-glucanase showed better germination performance against a wide range of temperatures and abscisic acid (ABA) stress. This can be attributed to the accumulation of Ph-glucanase at both transcript and protein levels during the seed germination in transgenic Arabidopsis. Results confirm that the cloned novel seed specific glucanase from a cold desert plant Podophyllum could be used for the manipulation of different plant species seeds against various harsh conditions.

Re-analysis of protein data reveals the germination pathway and up accumulation mechanism of cell wall hydrolases during the radicle protrusion step of seed germination in Podophyllum hexandrum- a high altitude plant.

Podophyllum hexandrum Royle is an important high-altitude plant of Himalayas with immense medicinal value. Earlier, it was reported that the cell wall hydrolases were up accumulated during radicle protrusion step of Podophyllum seed germination. In the present study, Podophyllum seed Germination protein interaction Network (PGN) was constructed by using the differentially accumulated protein (DAP) data set of Podophyllum during the radicle protrusion step of seed germination, with reference to Arabidopsis protein-protein interaction network (AtPIN). The developed PGN is comprised of a giant cluster with 1028 proteins having 10,519 interactions and a few small clusters with relevant gene ontological signatures. In this analysis, a germination pathway related cluster which is also central to the topology and information dynamics of PGN was obtained with a set of 60 key proteins. Among these, eight proteins which are known to be involved in signaling, metabolism, protein modification, cell wall modification, and cell cycle regulation processes were found commonly highlighted in both the proteomic and interactome analysis. The systems-level analysis of PGN identified the key proteins involved in radicle protrusion step of seed germination in Podophyllum.

Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea.

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals (O2( ·-)) to molecular oxygen (O2) and hydrogen peroxide (H2O2). Previously, we have identified and characterized a thermo-tolerant copper-zinc superoxide dismutase from Potentilla atrosanguinea (PaSOD), which retains its activity in the presence of NaCl. In the present study, we show that cotyledonary explants of PaSOD overexpressing transgenic Arabidopsis thaliana exhibit early callus induction and high shoot regenerative capacity than wild-type (WT) explants. Growth kinetic studies showed that transgenic lines have 2.6-3.3-folds higher growth rate of calli compared to WT. Regeneration frequency of calli developed from transgenic cotyledons was found to be 1.5-2.5-folds higher than that of WT explants on Murashige and Skoog medium supplemented with different concentrations of naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) within 2 weeks. A positive regulatory effect of PaSOD and H2O2 was observed on different stages of callusing and regeneration. However, this effect was more pronounced at the early stages of the regeneration processes in transgenic lines as compared to WT. These results clearly indicate that plant regeneration is regulated by endogenous H2O2 and by factors, which enhance its accumulation. Transgenics also exhibited salt stress tolerance with higher SOD activity, chlorophyll content, total soluble sugars, and proline content, while lower ion leakage and less reduction in relative water content, as compared to WT. Thus, it appears that the activation of PaSOD at regeneration stage accompanied by increased H2O2 production can be one of the mechanisms controlling in vitro morphogenesis.

Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

Protein changes during ethanol induced seed germination in Aconitum heterophyllum.

Aconitum heterophyllum is a high altitude medicinal plant that has become endangered due to overexploitation for their aconitins. The most effective, conventional propagation method for any plant species is by seed. However, in Aconitum seed germination is erratic, and seedling survival is low. In the present study results have been discussed on the possible implication of ethanol treatment on removal of barriers on radical emergence in terms of protein changes. Eighty seven percent of seed germination was achieved in Aconitum with ethanol treatment. Comparative 2-DE analysis of ethanol treated and untreated seed protein profiles in Phase II of germination revealed 40 differentially expressed proteins. Twenty-seven out of 40 proteins were induced, 5 were increased and 8 were repressed. Mass spectrometry and subsequent identification confirmed that these proteins were involved in metabolism, DNA regulation, stress tolerance and plasmamembrane/cell wall biosynthesis/extension processes. These protein changes might be responsible for physiological and physical changes, respectively, resulted in increase in germination percentage. Further, characterization of these proteins will be of great help in understanding the molecular mechanism lying behind enhanced germination in response to ethanol treatment.

Change in protein content during seed germination of a high altitude plant Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle (=Sinopodophyllum hexandrum) is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. An effective, conventional propagation method is by seed. However, seed germination is erratic, and seedling survival is low. A marginal increase in Podophyllum seed germination was attained with organic solvents. In the present study an attempt was made to decipher the physiological and biochemical barriers in terms of change in proteins during seed germination of Podophyllum. Comparative 2-DE analysis between un-germinated (dormant) and germinating seeds revealed nearly 113 differentially expressed proteins, whereas Peptide Mass Fingerprint (PMF) analysis of 97 protein spots revealed appearance of 27 proteins, up-accumulation of 11 proteins, down-accumulation of 19 proteins and disappearance of 40 proteins with germination. Identified 59 proteins in the homology search were involved in metabolism (carbohydrate and amino acid metabolism; 20 proteins), ABA/GA signaling (17 proteins) and stress (15 proteins) related proteins. Seven proteins were with unknown function. Two-DE, and MS/MS analysis in conjunction with semi-quantitative RT-PCR data of cell wall hydrolyzing genes, revealed that in Podophyllum the radicle protrusion occurs might be because of the up-accumulation of cell wall hydrolases i.e. ß-1, 3-glucanase and XET which weakens the thick walled micropylar endosperm.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Over-expression of superoxide dismutase exhibits lignification of vascular structures in Arabidopsis thaliana.

The present study demonstrated that over-expression of copper-zinc superoxide dismutase (Cu/Zn-SOD), an important enzyme scavenging reactive oxygen species, improved vascular structures through lignification and imparted tolerance to salt stress (NaCl) in Arabidopsis thaliana (Arabidopsis; accession Col-0). Transgenic plants of Arabidopsis were developed by over-expressing cytosolic Cu/Zn-SOD from Potentilla atrosanguinea under CaMV35S promoter via Agrobacterium tumefaciens mediated transformation. Homozygous T(3) lines were analyzed for morphological, anatomical and molecular differences in response to salt stress. The transgenic plants showed higher germination and survival percentage, larger root length, larger rosette area and the higher number of leaves as compared to the wild type (WT) under NaCl stress. Anatomical studies of the inflorescence stem revealed significant thickening of inter-vesicular cambium in transgenics under NaCl stress as compared to the (i) WT and the transgenics raised in the absence of NaCl stress, as well as (ii) WT raised under NaCl stress. This thickening was possibly due to lignification as evidenced by the confocal microscopy. Also, the up-regulation of transcripts of critical genes of lignin biosynthesis, phenylalanine ammonia-lyase1 (PAL1) and peroxidase (PRXR9GE) in the transgenics supported lignification of vascular tissue under the above stated conditions. Results have been discussed on the possible implication of over-expression of PaSOD in lignification of vascular structure under NaCl stress in Arabidopsis.