Jas Singh

Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus

Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5′-proximal region of the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.

Regulation and characterization of four CBF transcription factors from Brassica napus

Four orthologues of the Arabidopsis CBF/Dreb transcriptional activator genes were isolated from the winter Brassicanapus, cv. Jet neuf. All four BNCBF clones encode a putative DRE/CRT (LTRE)-binding protein with an AP2 DNA-binding domain, a putative nuclear localization signal and a possible acidic activation domain. Deduced amino acid sequences suggested that BNCBFs5, 7and 16 are very similar to the ArabidopsisCBF1 whereas BNCBF17 is different in that it contains two extra regions of 16 and 21 amino acids in the acidic domain. Transcripts hybridizing specifically to BNCBF17 and to one or more of the other BNCBFs accumulated in leaves within 30 min of cold exposure of the Brassica seedlings and preceded transcript accumulation of the cold-inducible BN28 gene, a Brassica orthologue of the cor6.6 or KIN gene from Arabidopsis. Cold-induced accumulation of BNCBF17 mRNA was rapid but was short-lived compared to transcripts hybridizing to BNCBF5/7/16. Transcripts hybridizing to one or more of BNCBF5/7/16 accumulated at low levels after the plants were subjected to prolonged exposure to salt stress. BNCBF17 was not responsive to salt stress. BNCBF transcript accumulation was similar in both spring and winter Brassica but the persistence of the transcripts in the cold were generally shorter in the spring than in the winter type. BNCBF5 and 17 proteins bind invitro to the LTRE domains of the cold-inducible BN115 (cor15a orthologue) or BN28 promoters. Differential binding preferences, however, to LTREs between BN115 and BN28 were observed. Mutation of the core CCGAC sequence of the LTRE indicated that BNCBF17 had a lower sequence binding specificity than BNCBF5. Furthermore, experiments indicated that the LTREs were able to drive BNCBF5 and 17trans-activation of the Lac-Z reporter gene in yeast. We conclude that the BNCBFs reported here could function as trans-acting factors in low-temperature responses in Brassica, controlling the expression of cold-induced genes through an ABA-independent pathway.

Induction of Freezing Tolerance in an Embryogenic Cell Suspension Culture of Brassica napus by Abscisic Acid at Room Temperature

Summary ABA was effective in circumventing the requirements for both low temperature and lengthy acclimation periods in the induction of freezing tolerance in an embryogenic microspore-derived cell suspension culture of winter rape (Brassica napus cv. Jet neuf). A higher level of freezing tolerance was induced when cells were cultured for 7 – 8 days in 5 × 10–5 M ABA, and 13 sucrose at 25 °C (60 – 70 % survival at −20 °C) then when cells were cultured in the same medium for 4 weeks at 2 °C (40 % survival at −20 °C). Acclimated cells which survived freezing at −20 °C retained their ability to undergo somatic embryogenesis.

Characterization of Three Related Low-Temperature-Regulated cDNAs from Winter Brassica napus

A cDNA clone, pBN115, encoding a low-temperature-regulated transcript in winter Brassica napus has been isolated. Northern blot analyses show that levels of transcripts hybridizing to pBN115 increase within 24 h of exposure of B. napus to low temperature, peak at 3 d, and then remain at an elevated level for the duration of the cold treatment (up to 10 weeks). Transferring plants from 2[deg]C to room temperature results in the loss of detectable transcripts hybridizing to pBN115 within 1 d. The transcript was not detected in RNA isolated from roots of cold-acclimated B. napus. Results of in vivo labeling of nascent RNA in leaf discs of B. napus with thiouridine suggest that regulation of expression may be transcriptional, at least at the onset of cold temperature. Although drought stress leads to a slight increase in transcript level at room temperature, neither a brief exposure to elevated temperatures nor exogenous application of abscisic acid resulted in the appearance of the transcript represented by pBN115. Furthermore, transcripts hybridizing to pBN115 were present at the same levels whether the plants were acclimated in the light or dark. Hybridization experiments show that pBN115 hybridizes strongly to cold-regulated transcripts in Arabidopsis thaliana, Descurania sophia, and spring B. napus, all of which are cruciferous plants capable of cold acclimation. No hybridizing transcript could be detected in cold-acclimated Spinacea oleracea, winter Secale cereale, or cold-grown Nicotiana tabacum. DNA sequence analysis of pBN115 reveals a single open reading frame that potentially encodes a protein of 14.8 kD. This size closely approximates that of a polypeptide produced by in vitro transcription/translation experiments. Two additional cDNA clones, pBN19 and pBN26, with divergent 5[prime]- and 3[prime]-untranslated regions, were also isolated and found to encode similar, but not identical, polypeptides.

Role of CBFs as Integrators of Chloroplast Redox, Phytochrome and Plant Hormone Signaling during Cold Acclimation

Cold acclimation of winter cereals and other winter hardy species is a prerequisite to increase subsequent freezing tolerance. Low temperatures upregulate the expression of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) which in turn induce the expression of COLD-REGULATED (COR) genes. We summarize evidence which indicates that the integration of these interactions is responsible for the dwarf phenotype and enhanced photosynthetic performance associated with cold-acclimated and CBF-overexpressing plants. Plants overexpressing CBFs but grown at warm temperatures mimic the cold-tolerant, dwarf, compact phenotype; increased photosynthetic performance; and biomass accumulation typically associated with cold-acclimated plants. In this review, we propose a model whereby the cold acclimation signal is perceived by plants through an integration of low temperature and changes in light intensity, as well as changes in light quality. Such integration leads to the activation of the CBF-regulon and subsequent upregulation of COR gene and GA 2-oxidase (GA2ox) expression which results in a dwarf phenotype coupled with increased freezing tolerance and enhanced photosynthetic performance. We conclude that, due to their photoautotrophic nature, plants do not rely on a single low temperature sensor, but integrate changes in light intensity, light quality, and membrane viscosity in order to establish the cold-acclimated state. CBFs appear to act as master regulators of these interconnecting sensing/signaling pathways.

Regulation of BN115, a Low-Temperature-Responsive Gene from Winter Brassica napus

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5[prime] regulatory region (from bp -1107 to +100) was fused to the [beta]-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2[deg]C. No expression was observed after incubation at 22[deg]C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5[prime] end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.

Characterization of a low-temperature-induced cDNA from winter Brassica napus encoding the 70 kDa subunit of tonoplast ATPase

A cDNA clone, pBN59, was isolated by differential screening of a cDNA library of winter Brassica napus during cold acclimation. Nucleotide sequence of BN59 was found to be homologous to that encoding the 70 kDa subunit of the vacuolar H+-ATPase in plants. Transcripts hybridizing to BN59 accumulated during exposure to low temperatures and to the exogenous application of abscisic acid (ABA). Western blot analyses also indicated an increase in the 70 kDa subunit during cold acclimation. The accumulation of an endomembrane H+-ATPase is consistent with the observation of osmotic adjustment, increases in endogenous ABA and the proliferation of endomembranes during cold acclimation.

Potential for increased photosynthetic performance and crop productivity in response to climate change: role of CBFs and gibberellic acid

We propose that targeting the enhanced photosynthetic performance associated with the cold acclimation of winter cultivars of rye (Secale cereale L.), wheat (Triticum aestivum L.), and Brassica napus L. may provide a novel approach to improve crop productivity under abiotic as well as biotic stress conditions. In support of this hypothesis, we provide the physiological, biochemical, and molecular evidence that the dwarf phenotype induced by cold acclimation is coupled to significant enhancement in photosynthetic performance, resistance to photoinhibition, and a decreased dependence on photoprotection through non-photochemical quenching which result in enhanced biomass production and ultimately increased seed yield. These system-wide changes at the levels of phenotype, physiology, and biochemistry appear to be governed by the family of C-repeat/dehydration-responsive family of transcription factors (CBF/DREB1). We relate this phenomenon to the semi-dwarf, gibberellic acid insensitive (GAI), cereal varieties developed during the “green revolution” of the early 1960s and 1970s. We suggest that genetic manipulation of the family of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) may provide a novel approach for the maintenance and perhaps even the enhancement of plant productivity under conditions of sub-optimal growth conditions predicted for our future climate.

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection.

Genome-wide analysis of drought induced gene expression changes in flax (Linum usitatissimum)

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.