Kamal Malik

Animal cell-death suppressors Bcl-xL and Ced-9 inhibit cell death in tobacco plants

In plants, events similar to programmed cell death have been reported [1] [2], although little is known of their mechanisms at the molecular level. To investigate the mechanism(s) involved, we overexpressed bcl-x(L), which encodes a mammalian suppressor of programmed cell death, in tobacco plants, under the control of a strong promoter [3]. In plants expressing Bcl-x(L), cell death induced by UV-B irradiation, paraquat treatment or the hypersensitive reaction (HR) to tobacco mosaic virus (TMV) infection was suppressed. The extent of suppression of cell death depended on the amount of Bcl-x(L) protein expressed. Similar enhanced resistance to cell death was found in transgenic tobacco plants overexpressing the ced-9 gene, a Caenorhabditis elegans homolog of bcl-x(L) [4], indicating that Bcl-x(L) and Ced-9 can function to inhibit cell death in plants.

Functional analysis of a RPD3 histone deacetylase homologue in Arabidopsis thaliana.

Histone acetylation is modulated through the action of histone acetyltransferase and deacetylase, which play key roles in the regulation of eukaryotic gene expression. We have screened the expressed sequence tag database with the yeast histone deacetylase RPD3 sequence and identified two Arabidopsis homologues, AtRPD3A and AtRPD3B. The deduced amino acid sequences of AtRPD3A and AtRPD3B show high overall homology (55% identity) to each other. AtRPD3A encodes a putative protein of 502 amino acids with 49% identity to the yeast RPD3. AtRPD3B encodes a putative protein of 471 amino acids and shares 55% amino acid identity with the yeast RPD3. Northern analysis indicated that AtRPD3A was highly expressed in the leaves, stems, flowers and young siliques of Arabidopsis plants, whereas the AtRPD3B transcript was not detected in these organs. An AtRPD3A fusion protein repressed transcription when directed to a promoter driving a reporter gene, indicating a role for AtRPD3A protein in gene repression. Arabidopsis plants were transformed with a gene construct comprising a truncated AtRPD3A cDNA in the antisense orientation driven by a strong constitutive promoter, −394tCUP. Antisense expression of AtRPD3A resulted in decreased endogenous AtRPD3A transcript and delayed flowering in transgenic Arabidopsis plants, suggesting that the transition from the vegetative to reproductive phase of development could be affected by histone acetylation. Our study demonstrates the important role of histone deacetylases in plant growth and development.

Ectopic Expression of an Arabidopsis Calmodulin-Like Domain Protein Kinase-Enhanced NADPH Oxidase Activity and Oxidative Burst in Tomato Protoplasts

Among plant defense responses to pathogen attack, the release of active oxygen species (AOS), termed the oxidative burst, may affect the attacking pathogen and the host plant cells at the infection site, thereby limiting the spread of the pathogen. Plasma membrane-associated NADPH oxidase represents a key enzyme in mediating the oxidative burst. The mechanisms of NADPH oxidase activation, however, remains unclear. Ectopic expression of AK1-6H, an Arabidopsis calmodulin-like domain protein kinase (CDPK) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity. Arabidopsis protein phosphatase 2A abolished this enhancement, whereas Arabidopsis dual-specificity protein tyrosine phosphatase 1 or maize protein phosphatase 1 had no effect tMEK2MUT, a constitutively activated, mitogen-activated protein kinase kinase from tomato, did not enhance NADPH oxidase activity when overexpressed. In a cell-free system, AK1-6H moderately stimulated the NADPH oxidase activity on plasma membrane. AK1-6H, but not tMEK2MUT, also enhanced production of AOS in intact protoplasts. Our results show that ectopic expression of a heterologous CDPK can enhance NADPH oxidase activity and stimulate an oxidative burst in tomato protoplasts.

A constitutive gene expression system derived from the tCUP cryptic promoter elements

Abstract.A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops (Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5′ untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a β-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed

Enhancers and core promoter elements are essential for the activity of a cryptic gene activation sequence from tobacco, tCUP.

Abstract. Cryptic gene regulatory elements are sequences that are inactive at their native locations in the genome but have the ability to become functional when positioned adjacent to genes. We have recently isolated such a cryptic sequence from tobacco, tCUP, that can act as a promoter. A 135-bp fragment spanning extending from position –197 to –62, relative to the transcription start site, was found to promote GUS expression in all of the major organs of transgenic Arabidopsis plants. Furthermore, this 135-bp fragment complemented the –46 minimal promoter of CaMV 35S and conferred constitutive expression on transgenic Arabidopsis plants. An electrophoretic mobility-shift assay showed that nuclear proteins prepared from tobacco leaves interact with the 135-bp fragment. tCUP has a core promoter that lacks the TATA consensus sequence but addition of a TATA-box sequence increased the core promoter activity by three-fold. The sequence surrounding the transcription start site of tCUP has sequence similarity with the initiator element (Inr), and deletion of this sequence significantly reduced promoter activity, suggesting that an essential Inr element may exist in the tCUP core promoter. Fusion of the GCC-box enhancer element from pathogenesis-related genes to the core promoter elevated tCUP core promoter activity. Our study indicates that cryptic promoters are similar in composition and organization to promoters associated with expressed genes and that their promoter elements can be combined to create composite promoters that are fully functional. This data provides direct evidence that the expression pattern of plant genes can be influenced by cryptic gene regulatory elements when they are brought into juxtaposition with genes through DNA rearrangements.

MALDI-Qq-TOF-MS and transient gene expression analysis indicated co-enhancement of β-1,3-glucanase and endochitinase by tMEK2 and the involvement of divergent pathways

Abstract A mitogen-activated protein kinase (MAPK) pathway has been demonstrated as a key pathway in plant defense against pathogen attacks. With proteomics approaches, we specifically studied activation events downstream of a MAPK kinase, tMEK2, in tomato. Overexpression of a constitutively activated tomato MAPK kinase gene ( tMEK2 MUT ) enhanced resistance of transgenic tomato lines to the virulent bacterial pathogen Pseudomonas syringae pv. tomato . Pathogenesis-related genes, PR1b1 , β-1,3-glucanase, and endochitinase were up-regulated by tMEK2 MUT . Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight-mass spectrometry analysis of total soluble leaf proteins indicated that β-1,3-glucanase and endochitinase are among the up-regulated proteins in these transgenic plants. Co-expression studies using a transient gene expression system have indicated that β-1,3-glucanase and endochitinase genes up-regulated by tMEK2 MUT were down-regulated by different specific phosphatases through dephosphorylation of certain downstream signaling molecules. Our observations indicate that increased products of β-1,3-glucanase and endochitinase genes downstream of tMEK2 may play an important role in achieving disease resistance.