Temperature-sensitive male sterility in rice determined by the roles of AGO1d in reproductive phasiRNA biogenesis and function.

Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial-temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive. Here, through histological and high-throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis. AGO1d preferentially binds to miR2118-triggered 21-nucleotide (nt) phasiRNAs with a 5'-terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21- and 24-nt phasiRNAs and temperature-sensitive male sterility. At lower temperatures, anthers of the ago1d mutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism. These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

Factors affecting the rapid changes of protein under short-term heat stress.

BACKGROUND: Protein content determines the state of cells. The variation in protein abundance is crucial when organisms are in the early stages of heat stress, but the reasons affecting their changes are largely unknown. RESULTS: We quantified 47,535 mRNAs and 3742 proteins in the filling grains of wheat in two different thermal environments. The impact of mRNA abundance and sequence features involved in protein translation and degradation on protein expression was evaluated by regression analysis. Transcription, codon usage and amino acid frequency were the main drivers of changes in protein expression under heat stress, and their combined contribution explains 58.2 and 66.4% of the protein variation at 30 and 40 °C (20 °C as control), respectively. Transcription contributes more to alterations in protein content at 40 °C (31%) than at 30 °C (6%). Furthermore, the usage of codon AAG may be closely related to the rapid alteration of proteins under heat stress. The contributions of AAG were 24 and 13% at 30 and 40 °C, respectively. CONCLUSION: In this study, we analyzed the factors affecting the changes in protein expression in the early stage of heat stress and evaluated their influence.

Metabolic adaptation of wheat grain contributes to a stable filling rate under heat stress.

Wheat (Triticum aestivum) is particularly vulnerable to heat stress during the grain filling stage, and this can adversely affect the final yield. However, the underlying physiological and molecular mechanisms are largely unknown. In this study, the effects of heat stress on grain filling were investigated using wheat varieties with different levels of thermotolerance. Decreased grain weights and filling durations, increased protein contents, and stable filling rates across diverse varieties under different heat regimes suggested a general mechanism for heat adaptation. Proteomic analysis identified 309 heat-responsive proteins (HRPs), and revealed a general decrease in protein synthesis components and metabolic proteins, but a significant increase in stress-response proteins and storage proteins. Metabolomic analysis identified 98 metabolites specifically changed by heat stress, and suggested a global decrease in the content of carbohydrate metabolites, an increased content of amino acids, and stable levels of starch synthesis precursors. The energy-consuming HRPs suggested that less energy was channelled into metabolism and protein synthesis, whereas more energy was allocated to the stress response under elevated heat conditions. Collectively, the data demonstrated a widely distributed mechanism for heat adaptation of metabolism, in which the assimilation and energy required for metabolism and protein synthesis are reallocated to heat protection and deposition of reserves, resulting in increased storage protein accumulation and a stable filling rate.

Downregulation of NADPH-cytochrome P450 reductase via RNA interference increases the susceptibility of Acyrthosiphon pisum to desiccation and insecticides.

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is involved in the metabolism of endogenous and exogenous substances, and detoxification of insecticides. RNA interference (RNAi) of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides. However, the CPR of Acyrthosiphon pisum has not been characterized, and its function is still not understood. In this study, we investigated the biochemical functions of A. pisum CPR (ApCPR). ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage, and in the gut, head, and abdominal cuticle. After optimizing the dose and silencing duration of RNAi for downregulating ApCPR, we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents, and of cuticular waxy coatings. Deficiency in cuticular hydrocarbons (CHCs) decreased the survival rate of A. pisum under desiccation stress and increased its susceptibility to contact insecticides. Moreover, desiccation stress induced a significant increase in ApCPR mRNA levels. We further confirmed that ApCPR participates in CHC production. These results indicate that ApCPR modulates CHC production, desiccation tolerance, and insecticide susceptibility in A. pisum, and presents a novel target for pest control.

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum.

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle. Lipophorin loads and transports lipids, including hydrocarbons, from one tissue to another. In some insects, the lipophorin receptor (LpR), which binds to lipophorin and accepts its lipid cargo, is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes. However, it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown. We herein present the results of our molecular characterization, phylogenetic analysis, and functional annotation of the pea aphid (Acyrthosiphon pisum) LpR gene (ApLpR). This gene was transcribed throughout the A. pisum life cycle, but especially during the embryonic stage and in the abdominal cuticle. Furthermore, we optimized the RHA interference (RNAi) parameters by determining the ideal dose and duration for gene silencing in the pea aphid. We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity. Additionally, a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress, with decreased survival rates under simulated drought conditions. Moreover, ApLpR expression levels significantly increased in response to the desiccation treatment. These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress. Furthermore, this gene is vital for aphid reproduction. Therefore, the ApLpR gene of A. pisum may be a novel RNAi target relevant for insect pest management.

Involvement of apolipoprotein D in desiccation tolerance and adult fecundity of Acyrthosiphon pisum.

Apolipoprotein D (ApoD) is a lipocalin superfamily member that plays important roles in the transport of small hydrophobic molecules, lipid metabolism, and stress resistance. Cuticular hydrocarbons are the principal components of the epicuticular lipid layer and play a critical role in water retention against environmental desiccation stress; however, the mechanism underlying the role of ApoD in insect desiccation tolerance has not yet been elucidated. Here, we report the molecular constitution, functional analysis, and phylogenetic relationship of the ApoD gene in Acyrthosiphon pisum (ApApoD). We found that ApApoD was transcribed throughout the life cycle of A. pisum, but was prominently expressed in the embryonic period and abdominal cuticle. In addition, we optimized the dose and silencing duration of RNAi, observing that RNAi against ApApoD significantly reduced the levels of both internal and cuticular hydrocarbons and adult fecundity. Moreover, cuticular hydrocarbon deficiency increased the sensitivity of aphids to desiccation stress and reduced their survival time, while desiccation stress significantly increased ApApoD expression. Together, it is confirmed that ApApoD participates in regulating cuticular hydrocarbon content of aphids under desiccation stress and is crucial for aphid reproduction. Therefore, the ApApoD gene of A. pisum may be a potential target for RNAi-based insect pest control due to its involvement in cuticular hydrocarbon accumulation and reproduction.

Varying Architecture of Heat Shock Elements Contributes to Distinct Magnitudes of Target Gene Expression and Diverged Biological Pathways in Heat Stress Response of Bread Wheat.

The heat shock transcription factor (HSF) binds to cis-regulatory motifs known as heat shock elements (HSEs) to mediate the transcriptional response of HSF target genes. However, the HSF-HSEs interaction is not clearly understood. Using the newly released genome reference sequence of bread wheat, we identified 39,478 HSEs (95.6% of which were non-canonical HSEs) and collapsed them into 30,604 wheat genes, accounting for 27.6% wheat genes. Using the intensively heat-responsive transcriptomes of wheat, we demonstrated that canonical HSEs have a higher propensity to induce a response in the closest downstream genes than non-canonical HSEs. However, the response magnitude induced by non-canonical HSEs was comparable to that induced by canonical HSEs. Significantly, some non-canonical HSEs that contain mismatched nucleotides at specific positions within HSEs had a larger response magnitude than that of canonical HSEs. Consistently, most of the HSEs identified in the promoter regions of heat shock proteins were non-canonical HSEs, suggesting an important role for these non-canonical HSEs. Lastly, distinct diverged biological processes were observed between genes containing different HSE types, suggesting that sequence variation in HSEs plays a key role in the evolution of heat responses and adaptation. Our results provide a new perspective to understand the regulatory network underlying heat responses.