Shiv S. Verma

Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis

APETALA2 (AP2) transcription factors (TFs) play very important roles in plant growth and development and in defense response. Here, we report functional characterization of four AP2 TF family genes [(RAP2.6 (At1g43160), RAP2.6L (At5g13330), DREB 26 (At1g21910) and DREB19 (At2g38340)] that were identified among NaCl inducible transcripts in abscisic acid responsive 17 (ABR17) transgenic Arabidopsis in our previous microarray analyses. DREB19 and DREB26 function as transactivators and localize in the nucleus. All four genes were abundant in early vegetative and flowering stages, although the magnitude of the expression varied. We observed tissue specific expression patterns for RAP2.6, RAP2.6L, DREB19 and DREB26 in flowers and other organs. RAP2.6 and RAP2.6L were responsive to stress hormones like jasmonic acid, salicylic acid, abscisic acid and ethylene in addition to salt and drought. DREB19 and DREB26 were less responsive to stress hormones, but the former was highly responsive to salt, heat and drought. Overexpression of RAP2.6 in Arabidopsis resulted in a dwarf phenotype with extensive secondary branching and small siliques, and DREB26 overexpression resulted in deformed plants. However, overexpression of RAP2.6L and DREB19 enhanced performance under salt and drought stresses without affecting phenotype. In summary, we have demonstrated that RAP2.6, RAP2.6L, DREB26 and DREB19 are transactivators, they exhibit tissue specific expression, and they participate in plant developmental processes as well as biotic and/or abiotic stress signaling. It is possible that the results from this study on these transcription factors, in particular RAP2.6L and DREB19, can be utilized in developing salt and drought tolerant plants in the future.

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.

Differential Expression of miRNAs in Brassica napus Root following Infection with Plasmodiophora brassicae

Canola (oilseed rape, Brassica napus L.) is susceptible to infection by the biotrophic protist Plasmodiophora brassicae, the causal agent of clubroot. To understand the roles of microRNAs (miRNAs) during the post-transcriptional regulation of disease initiation and progression, we have characterized the changes in miRNA expression profiles in canola roots during clubroot disease development and have compared these to uninfected roots. Two different stages of clubroot development were targeted in this miRNA profiling study: an early time of 10-dpi for disease initiation and a later 20-dpi, by which time the pathogen had colonized the roots (as evident by visible gall formation and histological observations). P. brassicae responsive miRNAs were identified and validated by qRT-PCR of miRNAs and the subsequent validation of the target mRNAs through starBase degradome analysis, and through 5′ RLM-RACE. This study identifies putative miRNA-regulated genes with roles during clubroot disease initiation and development. Putative target genes identified in this study included: transcription factors (TFs), hormone-related genes, as well as genes associated with plant stress response regulation such as cytokinin, auxin/ethylene response elements. The results of our study may assist in elucidating the role of miRNAs in post-transcriptional regulation of target genes during disease development and may contribute to the development of strategies to engineer durable resistance to this important phytopathogen.

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus)

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it’s in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot.

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.

A Simplified Floral Dip Method for Transformation of Brassica napus and B. carinata

A floral dip method of genetic transformation has been developed in two different species of Brassica namely B. napus cv Elect and i Pusa Gaurav. Binary vector containing B. carinata Zinc Finger transcription factor (BcZF1) gene under the transcriptional control of abiotic stress-inducible Late Embryogenesis Abundant 1 (i) promoter was transformed into Agrobacterium tumefaciens strain GV-3101. This strain was used for transformation of Brassica spp via Agrobacterium-mediated floral dip method. Agrobacterium cells cultured in Yeast Extract Mannitol (YEM) were harvested and resuspended in half MS solution containing 5% sucrose and 0.05% Silwet L-77. The transformation was performed by dipping inflorescence at the initial blossom stage. Seeds from these plants were harvested and screened by germinating them on MS medium supplemented with kanamycin (25 mg l−1). The kanamycin resistant putative transgenic plants were further confirmed by PCR and RT PCR using nptll gene-specific primers. The results showed that the Agrobacterium-mediated floral-dip transformation can be a successful strategy to develop transgenic B. napus and B. carinata in a short time without undergoing cumbersome vacuum infiltration or tissue culture steps.

miRNA-Mediated Posttranscriptional Regulation of Gene Expression in ABR17-Transgenic Arabidopsis thaliana Under Salt Stress

MicroRNAs (miRNAs) are a class of small endogenous RNAs conserved in eukaryotic organisms including plants. They suppress gene expression posttranscriptionally in many different biological processes. Previously, we reported salinity-induced changes in gene expression in transgenic Arabidopsis thaliana plants that constitutively expressed a pea abscisic acid-responsive (ABR17) gene. In the current study, we used microarrays to investigate the role of miRNA-mediated posttranscriptional gene regulation in these same transgenic plants in the presence and absence of salinity stress. We identified nine miRNAs that were significantly modulated due to ABR17 gene expression and seven miRNAs that were modulated in response to salt stress. The target genes regulated by these miRNAs were identified using sRNA target Base (starBase) degradome analysis and through 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Our findings revealed miRNA–mRNA interactions comprising regulatory networks of auxin response factor (ARF), argonaute 1 (AGO1), dicer-like proteins 1 (DCL1), SQUAMOSA promoter binding (SPB), NAC, APETALA 2 (AP2), nuclear factor Y (NFY), RNA-binding proteins, A. thaliana vacuolar phyrophosphate 1 (AVP1), and pentatricopeptide repeat (PPR) in ABR17-transgenic A. thaliana, which control physiological, biochemical, and stress signaling cascades due to the imposition of salt stress. Our results are discussed within the context of the effect of the transgene, ABR17, and the roles of miRNA expression may play in mediating plant responses to salinity.

Effects of lovastin, fosmidomycin and methyl jasmonate on andrographolide biosynthesis in the Andrographis paniculata

Andrographolide is a diterpene secondary metabolite product of Andrographis paniculata. It has been known to be a pharmaceutically important compound synthesized via the cytosolic mevalonate (MVA) and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. To understand the biosynthetic pathway of andrographolide biosynthesis in Andrographis paniculata, lovastatin, fosmidomycin and methyl jasmonate (MeJA) were used to inhibit the key enzymes 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), and 1-deoxy-d-xylulose-5-phosphate reducto-isomerase (DXR) involved in the synthesis of andrographolide in the MVA and MEP pathways, respectively. The inhibition of andrographolide accumulation was linked with the expression level of the studied regulatory genes, 3-hydroxy-3-methyl glutaryl coenzyme A synthase (hmgs), 3-hydroxy-3-methyl glutaryl coenzyme A reductase (hmgr), 1-deoxyxylulose-5-phosphate synthase (dxs), 1-deoxyxylulose-5-phosphate reductoisomerase (dxr), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (hds),1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (hdr), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase(isph), isopentenyl diphosphate isomerase (ipp), geranylgeranyl diphosphatesynthase (ggps) of the MVA and MEP pathways. The pathways associated transcript expression level, and andrographolide biosynthesis was significantly modulated by the inhibitors indicating that the andrographolide biosynthesis is strongly responsive at the transcriptional level. The results demonstrated that both pathways can contribute to the biosynthesis of andrographolide in A. paniculata. Both hmgr and dxr played a critical role consistent with some crossover between MVA and MEP pathways in andrographolide biosynthesis.

(E)-β-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines

Aphids are the major concern that significantly reduces the yield of crops. (E)-β-farnesene (Eβf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-β-farnesene (Eβf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eβf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-β-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using Eβf, a non-toxic mode of action.

APETALA2 Gene Family: Potential for Crop Improvement Under Adverse Conditions

Crop plants are exposed to many adverse conditions like biotic and abiotic stresses which affect their yield. Transcription factors which play important roles in the expression of stress-responsive genes, serve as valuable source for improving stress tolerance of crop plants. APETALA2/ethylene response element-binding protein (AP2/EREBP) transcription factor family is one of the major groups among the TF families in Arabidopsis. AP2 family members have been implicated in plant growth development as well as in stress signaling network. The DREB (dehydration-responsive element binding) subfamily proteins interact with C-repeat or dehydration response elements to regulate low-temperature and/or water deficit responsive genes, whereas, the ERF (ethylene-responsive factors) subfamily proteins interact with ethylene response elements (ERE) or GCC box to regulate the expression of pathogenesis-related genes. However, some DREB and ERF transcription factors are known to mediate cross talk between biotic and abiotic stress signaling pathways. Functional characterization of AP2 family genes has demonstrated usefulness of these genes in enhancing biotic/abiotic stress resistance/tolerance not only in model plants, but also in crop plants like rice, maize, wheat, barley, and soybean. Transgenic expression of a single AP2 TF has lead to improved tolerance to multiple stresses like salinity, drought, and heat stress and pathogen infection. Therefore engineering of AP2 TFs seems to be a valuable tool towards achieving enhanced crop productivity under adverse conditions.