Eva Czarnecka

AT-rich promoter elements of soybean heat shock gene Gmhsp 17.5E bind two distinct sets of nuclear proteins in vitro

A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (−907 to −889 and −843 to −826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5′ to a truncated (−140) maize Adh1 promoter. The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTi-based vector of Agrobacterium tumefaciens.Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5′ flanking regions of soybean β-conglycinin, lectin, leghemoglobin and heat shock genes. These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila. Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species. Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca. 46–69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins. The latter meets the operational criteria for high-mobility group proteins (HMGs).

Regulatory domains of the Gmhsp17.5-E heat shock promoter of soybean.

Promoter domains required for in vivo transcriptional expression of soybean heat shock gene Gmhsp17.5-E were identified by insertion-deletion mutagenesis with transgenic expression monitored in Agrobacterium tumefaciens-incited tumors of sunflower. Removal of the TATA-distal domain from position -1175 to position -259 had little effect on overall activity. The four regions contributing to promoter activity identified by this study all map within 244 base pairs from the start of transcription. The most distal cis-acting element of major significance was located from -244 to -179 and contains a conserved TATA-dyad motif centered at -220. Sequences from -179 to -40 comprise the TATA-proximal domain and include an AT-rich region and two sites containing heat shock consensus elements (HSEs). Deletion of the HSE centered at -93 (site 2) severely reduced transcriptional activity. Heat-inducible expression was also eliminated by internal deletion of either the TATA motif or the overlapping HSEs at site 1, indicating that each of these regions is also a major determinant of promoter activity.

Mutational analysis of a plant heat shock element

A total of 32 mutations were generated within the TATA-proximal site 1 (−72 to−47) of soybean heat shock gene Gmhsp17.5E in order to functionally define the optimal configuration of sequences within the heat shock element (HSE). Mutants were tested in vivo utilizing sunflower tumors transformed by a T-DNA based vector. Promoter activity was determined by S1 nuclease hybrid protection analysis of tumor transcripts. A total of five repeats (5′-nGAAn-3′ or 5′-nTTCn-3′) which comprise the HSE at site 1 were required for full transcription induction by heat stress. Analysis of non-conserved bases flanking the central trinucleotide block indicated that 5′-aGAAg′-3′ is the optimum sequence for the 5 bp repeat.

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants

We have designed and tested a transcriptional autofeedback loop that could be used to engineer plants to sense the presence of bacteria. The signal amplification circuit was built based on the biological switch responsive to the presence of bacterial flagellin. Several flagellin- and E. coli-inducible Arabidopsis promoters were cloned and tested in transient expression assays in Arabidopsis and lettuce protoplasts using a flagellin-based peptide. These were investigated either as direct drivers of a reporter gene, or as a component of a transcriptional autofeedback loop. Arabidopsis promoters from the xyloglucan endotransglucosylase/hydrolase 18 (ATXTH18) and cytochrome P450 family CYP82C3 monooxygenase worked well as biological switches. These promoters were incorporated into our feedback loop system for signal amplification. The inclusion of a transcriptional repressor reduced basal expression, thereby increasing fold-amplification of signal detection and fine-tuning the positive autofeedback loop regulation.

Feasibility of Using the Optical Sensing Techniques for Early Detection of Huanglongbing in Citrus Seedlings

A vision sensor was introduced and tested for early detection of citrus Huanglongbing (HLB). This disease is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllid. HLB is a devastating disease that has exerted a significant impact on citrus yield and quality in Florida. Unfortunately, no cure has been reported for HLB. Starch accumulates in HLB infected leaf chloroplasts, which causes the mottled blotchy green pattern. Starch rotates the polarization plane of light. A polarized imaging technique was used to detect the polarization-rotation caused by the hyper-accumulation of starch as a pre-symptomatic indication of HLB in young seedlings. Citrus seedlings were grown in a room with controlled conditions and exposed to intensive feeding by CLas-positive psyllids for eight weeks. A quantitative polymerase chain reaction was employed to confirm the HLB status of samples. Two datasets were acquired; the first created one month after the exposer to psyllids and the second two months later. The results showed that, with relatively unsophisticated imaging equipment, four levels of HLB infections could be detected with accuracies of 72%–81%. As expected, increasing the time interval between psyllid exposure and imaging increased the development of symptoms and, accordingly, improved the detection accuracy.

An Analysis of Physiological and Molecular Aspects of Heat Shock Gene Expression

The following discussion represents a brief summary of some aspects of the heat shock (HS) response, emphasizing observations made using soybean seedlings as the primary experimental system.