Minghui Lu

The Plant Heat Stress Transcription Factors (HSFs): Structure, Regulation, and Function in Response to Abiotic Stresses

Abiotic stresses such as high temperature, salinity, and drought adversely affect the survival, growth, and reproduction of plants. Plants respond to such unfavorable changes through developmental, physiological, and biochemical ways, and these responses require expression of stress-responsive genes, which are regulated by a network of transcription factors (TFs), including heat stress transcription factors (HSFs). HSFs play a crucial role in plants response to several abiotic stresses by regulating the expression of stress-responsive genes, such as heat shock proteins (Hsps). In this review, we describe the conserved structure of plant HSFs, the identification of HSF gene families from various plant species, their expression profiling under abiotic stress conditions, regulation at different levels and function in abiotic stresses. Despite plant HSFs share highly conserved structure, their remarkable diversification across plants reflects their numerous functions as well as their integration into the complex stress signaling and response networks, which can be employed in crop improvement strategies via biotechnological intervention.

Characterization of CaHsp70-1, a Pepper Heat-Shock Protein Gene in Response to Heat Stress and Some Regulation Exogenous Substances in Capsicum annuum L.

Pepper (Capsicum annuum L.) is sensitive to heat stress (HS). Heat shock proteins 70 (Hsp70s) play a crucial role in protecting plant cells against HS and control varies characters in different plants. However, CaHsp70-1 gene was not well characterized in pepper. In this study, CaHsp70-1 was cloned from the pepper thermotolerant line R9, which encoded a protein of 652 amino acids, with a molecular weight of 71.54 kDa and an isoelectric point of 5.20. CaHsp70-1 belongs to the cytosolic Hsp70 subgroup, and best matched with tomato SlHsp70. CaHsp70-1 was highly induced in root, stem, leaf and flower in R9 with HS treatment (40 °C for 2 h). In both thermosensitive line B6 and thermotolerant line R9, CaHsp70-1 significantly increased after 0.5 h of HS (40 °C), and maintained in a higher level after 4 h HS. The expression of CaHsp70-1 induced by CaCl2, H2O2 and putrescine (Put) under HS were difference between B6 and R9 lines. The different expression patterns may be related to the differences in promoters of CaHsp70-1 from the two lines. These results suggest that CaHsp70-1 as a member of cytosolic Hsp70 subgroup, may be involved in HS defense response via a signal transduction pathway contained Ca2+, H2O2 and Put.

Genome-wide analysis of the CaHsp20 gene family in pepper: comprehensive sequence and expression profile analysis under heat stress.

The Hsp20 genes are present in all plant species and play important roles in alleviating heat stress and enhancing plant thermotolerance by preventing the irreversible aggregation of denaturing proteins. However, very little is known about the CaHsp20 gene family in pepper (Capsicum annuum L.), an important vegetable crop with character of temperate but thermosensitive. In this study, a total of 35 putative pepper Hsp20 genes (CaHsp20s) were identified and renamed on the basis of their molecular weight, and then their gene structure, genome location, gene duplication, phylogenetic relationship, and interaction network were also analyzed. The expression patterns of CaHsp20 genes in four different tissues (root, stem, leaf, and flower) from the thermotolerant line R9 under heat stress condition were measured using semi-quantitative RT-PCR. The transcripts of most CaHsp20 genes maintained a low level in all of the four tissues under normal temperature condition, but were highly induced by heat stress, while the expression of CaHsp16.6b, 16.7, and 23.8 were only detected in specific tissues and were not so sensitive to heat stress like other CaHsp20 genes. In addition, compared to those in thermotolerant line R9, the expression peak of most CaHsp20 genes in thermosensitive line B6 under heat stress was hysteretic, and several CaHsp20 genes (CaHsp16.4, 18.2a, 18.7, 21.2, 22.0, 25.8, and 25.9) showed higher expression levels in both line B6 and R9. These data suggest that the CaHsp20 genes may be involved in heat stress and defense responses in pepper, which provides the basis for further functional analyses of CaHsp20s in the formation of pepper acquired thermotoleance.

Autophagy, a Conserved Mechanism for Protein Degradation, Responds to Heat, and Other Abiotic Stresses in Capsicum annuum L.

Abiotic stresses negatively affect plants growth and development by inducing protein denaturation, and autophagy degrades the damaged proteins to alleviate their toxicity, however, little is known about the involvement of autophagy in pepper (Capsicum annuum L.) tolerances to abiotic stresses. In this study, we identified autophagy-related gene (ATG) members in the whole genome of pepper by HMM method and analyzed their expression profiles in response to heat and other abiotic stresses by quantitative real-time PCR. The results showed that the CaATG contained 15 core ATG members including 29 ATG proteins with their respective conserved functional domains, involving the whole process of autophagy. Under normal environmental condition, the expression of CaATG genes showed tissue- and developmental stage-specific patterns, while under abiotic stresses of salt, drought, heat, cold and carbohydrate starvation, the accumulation of autophagosome punctate increased and the expression level of CaATG genes changed with stress type-dependent pattern, which indicates the linkage of autophagy in pepper response to abiotic stresses. After treated with heat stress, both the number of up-regulated CaATG genes and the increment of autophagosome punctate were higher in pepper thermotolerant line R9 than those in thermosensitive line B6, implying an association of autophagy with heat tolerance. In addition, CaATG6 was predicted to interact with CaHSP90 family members. Our study suggests that autophagy is connected to pepper tolerances to heat and other abiotic stresses.

Cloning and expression analysis of heat-shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L.).

The heat-shock transcription factor (Hsf) gene CaHsfA2 (GenBank accession No. JX402923) was cloned from the Capsicum annuum thermotolerant line R9 by combining the techniques electron cloning and rapid amplification of cDNA ends. The gene, which is 1436 bp in length, had an open reading frame of 1089 bp that encoded 362 amino acids. There was an 831-bp intron between positions 321 and 322 of the cDNA. The deduced amino acid sequence of CaHsfA2 contained the conserved domains of Hsf, including DNA binding domain, adjacent domain with heptad hydrophobic repeats (A/B), activator motifs, nuclear localization signal, and nuclear export signal, and it had the highest E value of hypothesized annotation of HsfA2. CaHsfA2 had the nearest phylogenetic relationship with HsfA2 from Lycopersicon peruvianum and Mimulus guttatus, which was consistent with its botanical classification. After heat-shock treatment at 40°C for 2 h, the expression of CaHsfA2 was observed in different tissues of thermotolerant cultivar R9 and thermosensitive line B6; however, the expression levels of the CaHsfA2 gene were significantly different as follows: expression in B6 leaf > stem > flower > root, and expression in R9 flower > leaf > stem ≈ root.

Characterization of BiP Genes from Pepper (Capsicum annuum L.) and the Role of CaBiP1 in Response to Endoplasmic Reticulum and Multiple Abiotic Stresses

Adverse environmental conditions have a detrimental impact on crop growth and development, and cause protein denaturation or misfolding. The binding protein (BiP) plays an important protective role by alleviating endoplasmic reticulum (ER) stress induced by misfolded proteins. In this study, we characterized three BiP genes (CaBiP1, CaBiP2, and CaBiP3) in pepper, an economically important vegetable and spice species. The role of CaBiP1 in plant tolerance to ER stress and adverse environmental conditions (including heat, salinity, osmotic and drought stress) were investigated. All the expected functional and signaling domains were detected in three BiP proteins, but the motifs and exon-intron distribution differed slightly in CaBiP3. CaBiP1 and CaBiP2 were constitutively expressed in all the tested tissues under both normal and stressed conditions, whereas CaBiP3 was mainly expressed following stress. Silencing of CaBiP1 reduced pepper tolerance to ER stress and various environment stresses, and was accompanied by increased H2O2 accumulation, MDA content, relative electric leakage (REL), water loss rate, and a reduction in soluble protein content and relative water content (RWC) in the leaves. Conversely, overexpression of CaBiP1 in Arabidopsis enhanced tolerance to ER stress and multiple environment stresses, as demonstrated by an increase in germination rate, root length, survival rate, RWC, the unfolded protein response (UPR) pathway, and a decrease in water loss rate. Our results suggest that CaBiP1 may contribute to plant tolerance to abiotic stresses by reducing ROS accumulation, increasing the water-retention ability, and stimulating UPR pathways and expression of stress-related genes.

Differential Responses to the Combined Stress of Heat and Phytophthora capsici Infection Between Resistant and Susceptible Germplasms of Pepper (Capsicum annuum L.)

Stresses caused by heat and Phytophthora capsici infection reduce pepper (Capsicum annuum) yields; however, little is known about their combined effects on pepper. In this study, the combination of heat and P. capsici infection resulted in worse symptoms, including increased heat stress injury, cell death, relative electrical conductivity, and decreased root activity, than either individual stress. The symptoms were more obvious in the susceptible pepper cultivar ‘Early Calwonder’ (EC) than in the resistant landrace line ‘Criollo de Morelos 334’ (CM334). Under heat stress alone, the expression levels of three tested C. annuum heat-shock protein (CaHSP) genes were enhanced in both ‘CM334’ and ‘EC’; however, under the combined stress, their expression levels increased in ‘CM334,’ but decreased in ‘EC.’ Among four defense-related genes, under stress treatments, only two were up-regulated in ‘CM334’ and ‘EC,’ respectively, and both showed higher levels under the combined stress than under the individual stresses. Under the combined stress, the expression levels of three tested Ca2+-signaling pathway genes were enhanced in ‘EC,’ but inhibited in ‘CM334’ compared with either heat or P. capsici infection. Compared with the effects caused by either heat or P. capsici infection, the CaWRKY expression levels were restored to nearly normal levels under the combined stress, and the number of up-regulated genes was higher in ‘CM334’ than in ‘EC.’ Thus, the molecular responses of pepper to the combined stress of heat and P. capsici infection are different from those caused by individual stresses, and the stresses interaction is different between resistant and susceptible pepper germplasms.