Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses.

Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress conditions like drought, salt, heat and cold stress adversely affect the productivity and quality of mulberry leaves. Quantitative real time PCR (qPCR) is a reliable and widely used method to identify abiotic stress responsive genes and molecular mechanism in different plant species. Selection of suitable reference genes is important requirement for normalizing the expression of genes through qRT-PCR study. In the present study, we have selected eight candidate reference genes in mulberry for analyzing their expression stability in different abiotic stress treatments including drought, salt, heat and cold stresses. The expression stability of these reference genes was determined using geNorm, NormFinder and RefFinder statistical algorithms. The results showed that Ubiquitin and protein phosphatase 2A regulatory subunit A (PP2A) were the most stable genes across all the treatment samples. However, analysis of individual stresses revealed different expression profiles and stability of reference genes. Actin3 and PP2A were most stable in drought and salt conditions respectively. RPL3 most preferred in heat stress and Ubiquitin was most stable in cold stress. We propose the ubiquitin and PP2A are the preferred reference genes for normalization of gene expression data from abiotic stresses. In addition, Actin3 are preferred for drought stress, PP2A for salt stress, RPL3 for heat stress and Ubiquitin for cold stress studies.

Transcriptomics and functional genomics of ROS-induced cell death regulation by RADICAL-INDUCED CELL DEATH1.

Plant responses to changes in environmental conditions are mediated by a network of signaling events leading to downstream responses, including changes in gene expression and activation of cell death programs. Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) has been proposed to regulate plant stress responses by protein-protein interactions with transcription factors. Furthermore, the rcd1 mutant has defective control of cell death in response to apoplastic reactive oxygen species (ROS). Combining transcriptomic and functional genomics approaches we first used microarray analysis in a time series to study changes in gene expression after apoplastic ROS treatment in rcd1. To identify a core set of cell death regulated genes, RCD1-regulated genes were clustered together with other array experiments from plants undergoing cell death or treated with various pathogens, plant hormones or other chemicals. Subsequently, selected rcd1 double mutants were constructed to further define the genetic requirements for the execution of apoplastic ROS induced cell death. Through the genetic analysis we identified WRKY70 and SGT1b as cell death regulators functioning downstream of RCD1 and show that quantitative rather than qualitative differences in gene expression related to cell death appeared to better explain the outcome. Allocation of plant energy to defenses diverts resources from growth. Recently, a plant response termed stress-induced morphogenic response (SIMR) was proposed to regulate the balance between defense and growth. Using a rcd1 double mutant collection we show that SIMR is mostly independent of the classical plant defense signaling pathways and that the redox balance is involved in development of SIMR.

RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana.

Transcriptional regulation of gene expression is one major determinant of developmental control and stress adaptation in virtually all living organisms. In recent years numerous transcription factors controlling various aspects of plant life have been identified. The activity of transcription factors needs to be regulated to prevent unspecific, prolonged or inappropriate responses. The transcription factor DREB2A (DEHYDRATION-RESPONSIVE ELEMENT BINDING 2A) has been identified as one of the main regulators of drought and heat responses, and it is regulated through protein stability. In the present paper we describe evidence that the interaction with RCD1 (RADICAL-INDUCED CELL DEATH 1) contributes to the control of DREB2A under a range of conditions. The interaction is mediated by a novel protein motif in DREB2A and a splice variant of DREB2A which lacks the interaction domain accumulates during heat stress and senescence. In addition RCD1 is rapidly degraded during heat stress, thus our results suggest that removal of RCD1 protein or the loss of the interaction domain in DREB2A appears to be required for proper DREB2A function under stress conditions.

Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.

RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the only Arabidopsis protein containing the WWE domain, which is known to mediate protein-protein interactions in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1 and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1 has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle - unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor collection revealed that RCD1 interacts with a large number of transcription factors belonging to several protein families, such as AP2/ERF, NAC and basic helix-loop-helix (bHLH), and that SRO1 interacts with a smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are required for proper plant development.

The RST and PARP-like domain containing SRO protein family: analysis of protein structure, function and conservation in land plants.

BACKGROUND: The SROs (SIMILAR TO RCD-ONE) are a group of plant-specific proteins which have important functions in stress adaptation and development. They contain the catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition to these domains, several, but not all, SROs contain an N-terminal WWE domain. RESULTS: SROs are present in all analyzed land plants and sequence analysis differentiates between two structurally distinct groups; cryptogams and monocots possess only group I SROs whereas eudicots also contain group II. Group I SROs possess an N-terminal WWE domain (PS50918) but the WWE domain is lacking in group II SROs. Group I domain structure is widely represented in organisms as distant as humans (for example, HsPARP11). We propose a unified nomenclature for the SRO family. The SROs are able to interact with transcription factors through the C-terminal RST domain but themselves are generally not regulated at the transcriptional level. The most conserved feature of the SROs is the catalytic core of the poly(ADP-ribose) polymerase (PS51059) domain. However, bioinformatic analysis of the SRO PARP domain fold-structure and biochemical assays of AtRCD1 suggested that SROs do not possess ADP-ribosyl transferase activity. CONCLUSIONS: The SROs are a highly conserved family of plant specific proteins. Sequence analysis of the RST domain implicates a highly preserved protein structure in that region. This might have implications for functional conservation. We suggest that, despite the presence of the catalytic core of the PARP domain, the SROs do not possess ADP-ribosyl transferase activity. Nevertheless, the function of SROs is critical for plants and might be related to transcription factor regulation and complex formation.

Biochemical and molecular analyses of copper-zinc superoxide dismutase from a C4 plant Pennisetum glaucum reveals an adaptive role in response to oxidative stress.

Superoxide dismutases (SODs) form the foremost line of defense against ROS in aerobes. Pennisetum glaucum cDNA library is constructed to isolate superoxide dismutase cDNA clone (PgCuZnSOD) of 798 bp comprising 5'UTR (111 bp), an ORF (459 bp) and 3'UTR (228 bp). Deduced protein of 152 amino acids (16.7 kDa) with an estimated isoelectric point of 5.76 shared highest homology to cytoplasmic CuZnSODs from monocots i.e., maize, rice. Predicted 3D model reveals a conserved eight-stranded ß-barrel with active site held between barrel and two surface loops. Purified recombinant protein is relatively thermo-stable with maximal activity at pH 7.6 and shows inhibition with H(2)O(2) (4.3 mM) but not with azide (10 mM). In Pennisetum seedlings, abiotic stress induced PgCuZnSOD transcript up-regulation directly correlates to high protein and activity induction. Overexpression of PgCuZnSOD confers comparatively enhanced tolerance to methyl viologen (MV) induced oxidative stress in bacteria. Results imply that PgCuZnSOD plays a functional role in conferring oxidative stress tolerance to prokaryotic system and may hold significant potential to impart oxidative stress tolerance in higher plants through transgenic approach.

Biochemical and molecular characterization of stress-induced ß-carbonic anhydrase from a C(4) plant, Pennisetum glaucum.

Genes encoding for many ß-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a ß-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C(4) crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant ß-CA family members with an over all conserved architecture of a typical ß-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast ß-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO(2) hydration and esterase activities. The ß-CAs studied so far possessed only CO(2) hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plant's adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO(2) supply within plant cells.

Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: interlinking oxidative and heat-stress responses.

The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.