Abstract
The analysis of stress-responsiveness in plants is an important route to
the discovery of genes conferring stress tolerance and their use in breeding
programs. High temperature is one of the environmental stress factors that
can affect the growth and quality characteristics of crops. The mechanisms
plants use to adapt to high temperature stress have been widely studied in a
number of plants. Major research has been focused on the isolation of heat
shock protein (HSP) genes as a means to understand the molecular events
underlying the adaptation process. Unfortunately, little/no attention has been
paid to identify high temperature stress-induced genes other than HSPs and
to understand the molecular aspects of response of hot pepper to high
temperature stress. Thus there is an urgent need to identify, clone and
characterize more high temperature stress-induced, previously
uncharacterized genes as well as transcription factors or regulatory proteins
for redesigning crops to better cope with abiotic stresses.
To enrich CDNA clones for regulatory proteins and transcription
factor/signal transduction component genes, we used modified differential
screening method, double negative screening approach. A cDNA library was
constructed for hot pepper plants that have been heat-shock-treated. From
the CDNA library, 500 CDNA clones representing genes with low expression
levels under high temperature stress were isolated by a modified differential
screening method, double negative screening. From the 500 cDNA clones, 200 cDNA clones were randomly selected and single read sequenced from the
5' ends. The sequence was annotated with blastx first, and then applied to
InterProScan to scan for functional motifs of proteins. Among the cDNA
clones analyzed, about 41% of the ESTs could not be functionally classified.
Among the functionally classified genes, the largest portion of ESTs, 15%,
were assigned to the category of cell rescue and defense category, while
genes involved in cell cycle/DNA processing constituted the smallest group,
comprising 1% of the ESTs. Genes involved in the energy and protein fate
formed the second (10%) and third (9%) largest groups, respectively. There
were 3% of ESTs assigned to the transcription, and 2% to the signal
transduction. The high portion of ESTs in the unclassified group was
probably resulted from the screening method designed for low expression
messages. Likewise, the high portion of ESTs in the cell rescue and defense
categories suggests that there are many genes with low expression levels in
the stress-related groups.
Through the use of differential hybridization screening, we have isolated
85 cDNA clones that are rapidly induced by high temperature stress in hot
pepper (Capsicum annuum L.). These clones were named as CaHTSI. Most
of the isolated clones were heat shock protein (HSPs). We have chosen 7
CDNA clones whose transcripts were induced strongly or slightly during heat
stress. These clones were searched for homology in the database, and four of
them were temporarily assigned for proteins as they showed significant
homologies to the proteins in amino acid sequences. Four clones encoded for
adenylate kinase, photosystem I reaction center subunit, PSI-D, Gram domain containing homolog/ABA responsive protein, Bax inhibitor-1. These proteins
have not been reported previously as high temperature stress-induced except
Gram domain containing homolog/ABA responsive protein although it is
evident that ABA responsive protein is stress inducible protein there has been
no report about the Gram domain containing homolog. Three cDNA clones
were novel with no significant homology with previously reported amino acid
sequences.
A hot pepper (Capsicum annuum L.) ring zinc finger protein gene,
CaRZFP, was isolated from a high temperature stress-induced cDNA library.
CaRZFP contains one conserved RING domain, and shows similarity with
Arabidopsis thaliana ring zinc finger protein in both overall amino-acid
sequences and the secondary structure arrangement within the conserved
DNA-binding motifs. The cDNA sequence contains an open reading frame of
657 bp, encoding a putative protein of 219 amino acid residues with a
predicted molecular mass of 23.75 kDa. Southern analysis indicated the
presence of a single-copy of the CaRZFP gene in the Capsicum annuum
genome. Northern analysis showed that CaRZFP is induced after different
types of stresses, namely high temperature, low temperature, drought, salt,
and but not by heavy metals. Accumulation of transcripts was not detected in
the control plants for all stresses. Thus, CaRZFP seems to be an important
determinant of stress response in plants.
In conclusion, double negative screening approach is suitable to isolate
transcription factors and signal transduction component genes as well as
regulatory proteins. It is also notable that proteins with unknown function or unclassified protein coding genes also increased by this approach. A high
temperature-induced cDNA clone CaRZFP was also induced by cold, drought,
and high salinity but not by heavy metals suggesting its potential utility to
improve stress tolerance in hot pepper plants.