Transgenic sugarcane with higher levels of BRK1 showed improved drought tolerance.

KEY MESSAGE: Transgenic sugarcane overexpressing BRK1 showed improved tolerance to drought stress through modulation of actin polymerization and formation of interlocking marginal lobes in epidermal leaf cells, a typical feature associated with BRK1 expression under drought stress. BRICK1 (BRK1) genes promote leaf epidermal cell morphogenesis and division in plants that involves local actin polymerization. Although the changes in actin filament organization during drought have been reported, the role of BRK in stress tolerance remains unknown. In our previous work, the drought-tolerant Erianthus arundinaceus exhibited high levels of the BRK gene expression under drought stress. Therefore, in the present study, the drought-responsive gene, BRK1 from Saccharum spontaneum, was transformed into sugarcane to test if it conferred drought tolerance in the commercial sugarcane cultivar Co 86032. The transgenic lines were subjected to drought stress, and analyzed using physiological parameters for drought stress. The drought-induced BRK1-overexpressing lines of sugarcane exhibited significantly higher transgene expression compared with the wild-type control and also showed improved physiological parameters. In addition, the formation of interlocking marginal lobes in the epidermal leaf cells, a typical feature associated with BRK1 expression, was observed in all transgenic BRK1 lines during drought stress. This is the first report to suggest that BRK1 plays a role in sugarcane acclimation to drought stress and may prove to be a potential candidate in genetic engineering of plants for enhanced biomass production under drought stress conditions.

Isolation and characterization of water-deficit stress-responsive α-expansin 1 (EXPA1) gene from Saccharum complex.

In this study, full-length (1282-1330 bp) α-expansin 1 (EXPA1) gene from three different accessions belonging to Saccharum complex (Saccharum officinarum-SoEXPA1, Erianthus arundinaceus-EaEXPA1, and Saccharum spp. hybrid-ShEXPA1) was isolated using RAGE technique and characterized. The intronic and coding regions of isolated expansin genes ranged between 526-568 and 756-762 bp, respectively. An open reading frame encoding a polypeptide of 252 amino acids was obtained from S. officinarum and commercial sugarcane hybrid, whereas 254 amino acids were obtained in E. arundinaceus, a wild relative of Saccharum. Bioinformatics analysis of deduced protein revealed the presence of specific signature sequences and conserved amino acid residues crucial for the functioning of the protein. The predicted physicochemical characterization showed that the protein is stable in nature with instability index (II) value less than 40 and also clearly shown the dominance of random coil in the protein structure. Phylogenetic analysis revealed high conservation of EXPA1 among Saccharum complex and related crop species, Sorghum bicolor and Zea mays. The docking study of EXPA1 protein showed the interaction with xylose, which is present in xyloglucan of plant cell wall, elucidated the role of the expansin proteins in plant cell wall modification. This was further supported by the subcellular localization experiment in which it is clearly seen that the expansin protein localizes in the cell wall. Relative expression analysis of EXPA1 gene in Saccharum complex during drought stress showed high expression of the EaEXPA1 in comparison with SoEXPA1 and ShEXPA1 indicating possible role of EaEXPA1 in increased water-deficit stress tolerance in E. arundinaceus. These results suggest the potential use of EXPA1 for increasing the water-deficient stress tolerance levels in crop plants.

A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems.

In genetic engineering, inducible promoters play an important role as the expression of genes driven by them can be turned on or off under situations like biotic or abiotic factors. There are few reports on inducible promoters that can be employed in the development of transgenic plants, particularly in sugarcane. In the present study, four wound inducible genes (Chitinase, PR1A, PR10 and HRGP) were selected and were amplified from Erianthus arundinaceus, a distant relative of sugarcane. In order to determine the gene that is highly induced upon wounding, RT-qPCR was performed, which showed that PR10 gene expression was instantaneous and higher upon wounding when compared to the other three genes. Using the random amplification of genomic ends technique, a 592 bp promoter sequence was obtained and in silico analysis of the upstream regulatory region revealed a 469 bp promoter and 123 bp of 5' untranslated region (UTR). Functional analyses of the promoter sequence (with and without 5' UTR) in tobacco, rice and sugarcane using ß-glucuronidase (GUS) as the reporter gene revealed the constitutive and inducible nature of the PR10 promoter. Our studies have demonstrated that the PR10 promoter, though highly constitutive, was quickly induced upon wounding as well as on treatment with abscisic acid and methyl jasmonate hormones. This is the first report on the isolation and characterization of a PR10 promoter from a wild grass and is expected to have application for development of transgenic plants.

Erianthus arundinaceus HSP70 (EaHSP70) Acts as a Key Regulator in the Formation of Anisotropic Interdigitation in Sugarcane (Saccharum spp. hybrid) in Response to Drought Stress.

Plant growth during abiotic stress is a long sought-after trait especially in crop plants in the context of global warming and climate change. Previous studies on leaf epidermal cells have revealed that during normal growth and development, adjacent cells interdigitate anisotropically to form cell morphological patterns known as interlocking marginal lobes (IMLs), involving the cell wall-cell membrane-cortical actin continuum. IMLs are growth-associated cell morphological changes in which auxin-binding protein (ABP), Rho GTPases and actin are known to play important roles. In the present study, we investigated the formation of IMLs under drought stress and found that Erianthus arundinaceus, a drought-tolerant wild relative of sugarcane, develops such growth-related cell morphological patterns under drought stress. Using confocal microscopy, we showed an increasing trend in cortical F-actin intensity in drought-tolerant plants with increasing soil moisture stress. In order to check the role of drought tolerance-related genes in IML formation under soil moisture stress, we adopted a structural data mining strategy and identified indirect connections between the ABPs and heat shock proteins (HSPs). Initial experimental evidence for this connection comes from the high transcript levels of HSP70 observed in drought-stressed Erianthus, which developed anisotropic interdigitation, i.e. IMLs. Subsequently, by overexpressing the E. arundinaceus HSP70 gene (EaHSP70) in sugarcane (Saccharum spp. hybrid), we confirm the role of HSP70 in the formation of anisotropic interdigitation under drought stress. Taken together, our results suggest that EaHSP70 acts as a key regulator in the formation of anisotropic interdigitation in drought-tolerant plants (Erianthus and HSP70 transgenic sugarcane) under moisture stress in an actin-mediated pathway. The possible biological significance of the formation of drought-associated interlocking marginal lobes (DaIMLs) in sugarcane plants upon drought stress is discussed.

Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.

Truncated Ubiquitin 5' Regulatory Region from Erianthus arundinaceus Drives Enhanced Transgene Expression in Heterologous Systems.

Characterization of novel plant gene promoters underpins the development of transgenic crop plants. Here, we report a novel 5' regulatory sequence (Eriubi D7) of the ubiquitin gene from Erianthus arundinaceus, a wild relative of sugarcane resistant to many biotic and abiotic stresses. A 3.2-kb regulatory sequence of ubiquitin gene was isolated through random amplification of genomic ends technique and characterized in rice, tobacco, and sugarcane. In silico analysis revealed that the regulatory sequence contained a promoter region of 1600 bp upstream to the transcription start site. Between the promoter and the coding region, two putative introns of 584 and 583 bp and two putative non-coding exons of 459 and 37 bp were spaced alternatively. To identify the active domains required for gene regulation, 12 truncations/recombinants were made in the regulatory sequence and characterized in heterologous systems. Transformation studies with the recombinant constructs revealed that Eriubi D7, a truncated fragment containing 830 bp promoter and the intron I, conferred enhanced GUS reporter gene expression in both monocots and dicots compared to other routinely used promoters such as maize ubi1 and Cauliflower Mosaic Virus 35S. Further analysis confirms that this regulatory sequence is quite distinct from the other reported ubiquitin promoters and was also found to enhance expression of the reporter gene upon wounding. This is the first report on the isolation and characterization of a promoter from a wild sugarcane germplasm and is expected to be useful for development of transgenic crop plants.

Introduction of Pea DNA Helicase 45 Into Sugarcane (Saccharum spp. Hybrid) Enhances Cell Membrane Thermostability And Upregulation Of Stress-responsive Genes Leads To Abiotic Stress Tolerance.

DNA helicases are motor proteins that play an essential role in nucleic acid metabolism, by providing a duplex-unwinding function. To improve the drought and salinity tolerance of sugarcane, a DEAD-box helicase gene isolated from pea with a constitutive promoter, Port Ubi 2.3 was transformed into the commercial sugarcane variety Co 86032 through Agrobacterium-mediated transformation, and the transgenics were screened for tolerance to soil moisture stress and salinity. The transgene integration was confirmed through polymerase chain reaction, and the V 0 transgenic events showed significantly higher cell membrane thermostability under normal irrigated conditions. The V 1 transgenic events were screened for tolerance to soil moisture stress and exhibited significantly higher cell membrane thermostability, transgene expression, relative water content, gas exchange parameters, chlorophyll content, and photosynthetic efficiency under soil moisture stress compared to wild-type (WT). The overexpression of PDH45 transgenic sugarcane also led to the upregulation of DREB2-induced downstream stress-related genes. The transgenic events demonstrated higher germination ability and better chlorophyll retention than WT under salinity stress. Our results suggest the possibility for development of increased abiotic stress tolerant sugarcane cultivars through overexpression of PDH45 gene. Perhaps this is the first report, which provides evidence for increased drought and salinity tolerance in sugarcane through overexpression of PDH45.

Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

Heat shock proteins (HSPs) have a major role in stress tolerance mechanisms in plants. Our studies have shown that the expression of HSP70 is enhanced under water stress in Erianthus arundinaceus. In this paper, we evaluate the effects of overexpression of EaHSP70 driven by Port Ubi 2.3 promoter in sugarcane. The transgenic events exhibit significantly higher gene expression, cell membrane thermostability, relative water content, gas exchange parameters, chlorophyll content and photosynthetic efficiency. The overexpression of EaHSP70 transgenic sugarcane led to the upregulation of stress-related genes. The transformed sugarcane plants had better chlorophyll retention and higher germination ability than control plants under salinity stress. Our results suggest that EaHSP70 plays an important role in sugarcane acclimation to drought and salinity stresses and its potential for genetic engineering of sugarcane for drought and salt tolerance.

Overexpression of EaDREB2 and pyramiding of EaDREB2 with the pea DNA helicase gene (PDH45) enhance drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

KEY MESSAGE: EaDREB2 overexpressed in sugarcane enhanced tolerance to drought and salinity. When co-transformed with plant DNA helicase gene, DREB2 showed greater level of salinity tolerance than in single-gene transgenics. Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and can potentially cause up to 50 % yield loss. DREB proteins play a vital regulatory role in abiotic stress tolerance in plants. We previously reported that expression of EaDREB2 is enhanced by drought stress in Erianthus arundinaceus. In this study, we have isolated the DREB2 gene from E. arundinaceus, transformed one of the most popular sugarcane variety Co 86032 in tropical India with EaDREB2 through Agrobacterium-mediated transformation, pyramided the EaDREB2 gene with the gene coding for PDH45 driven by Port Ubi 2.3 promoter through particle bombardment and evaluated the V1 transgenics for soil deficit moisture and salinity stresses. Soil moisture stress was imposed at the tillering phase by withholding irrigation. Physiological, molecular and morphological parameters were used to assess drought tolerance. Salinity tolerance was assessed through leaf disc senescence and bud sprout assays under salinity stress. Our results indicate that overexpression of EaDREB2 in sugarcane enhances drought and salinity tolerance to a greater extent than the untransformed control plants. This is the first report of the co-transformation of EaDREB2 and PDH45 which shows higher salinity tolerance but lower drought tolerance than EaDREB2 alone. The present study seems to suggest that, for combining drought and salinity tolerance together, co-transformation is a better approach.

5' Regulatory region of ubiquitin 2 gene from Porteresia coarctata makes efficient promoters for transgene expression in monocots and dicots.

KEY MESSAGE: Porteresia ubiquitin 5' regulatory region drives transgene expression in monocots and dicots. Ubiquitin promoters are promising candidates for constitutive transgene expression in plants. In this study, we isolated and characterized a novel 5' regulatory sequence of a ubiquitin gene from Porteresia coarctata, a stress-tolerant wild grass species. Through functional analysis in heterologous plant systems, we have demonstrated that full length (Port Ubi2.3) or truncated sequence (PD2) of the isolated regulatory fragment can drive constitutive expression of GUS in monocots and/or dicots. In silico analysis of Port Ubi2.3 has revealed the presence of a 640 bp core promoter region followed by two exons and two introns with numerous putative cis-acting sites scattered throughout the regulatory region. Transformation and expression studies of six different deletion constructs in rice, tobacco and sugarcane revealed that the proximal intron has an enhancing effect on the activity of the core promoter in both monocots and dicots, whereas, Port Ubi2.3 was able to render strong expression only in monocots. This regulatory sequence is quite distinct from the other reported ubiquitin promoters in structure and performs better in monocots compared to other commonly used promoters-maize Ubi1 and Cauliflower Mosaic Virus 35S.

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance.

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC(50) value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.

Engineering sugarcane cultivars with bovine pancreatic trypsin inhibitor (aprotinin) gene for protection against top borer (Scirpophaga excerptalis Walker).

The inhibitory activity of bovine pancreatic trypsin inhibitor (aprotinin), a natural polypeptide and a proteinase inhibitor, was demonstrated on gut proteinases of three lepidopteran borers of sugarcane using commercially available aprotinin. A synthetic gene coding for aprotinin, designed and codon optimized for better expression in plant system (Shantaram 1999), was transferred to two sugarcane cultivars namely CoC 92061 and Co 86032 through particle bombardment. Aprotinin gene expression was driven by maize ubiquitin promoter and the plant selection marker used was hygromycin resistance. The integration, expression and functionality of the transgene was confirmed by Southern, Western and insect bioassay, respectively. Southern analysis showed two to four integration sites of the transgene in the transformed plants. Independent transgenic events showed varied levels of transgene expression resulting in different levels (0.16-0.50%) of aprotinin. In in vivo bioassay studies, larvae of top borer Scirpophaga excerptalis Walker (Lepidoptera: Pyralidae) fed on transgenics showed significant reduction in larval weight which indicated impairment of their development. Results of this study show the possibility of deploying aprotinin gene for the development of transgenic sugarcane cultivars resistant to top borer.