Proteasome resides in and dismantles plant heat stress granules constitutively.

Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.

Phenolic acid-induced phase separation and translation inhibition mediate plant interspecific competition.

Phenolic acids (PAs) secreted by donor plants suppress the growth of their susceptible plant neighbours. However, how structurally diverse ensembles of PAs are perceived by plants to mediate interspecific competition remains a mystery. Here we show that a plant stress granule (SG) marker, RNA-BINDING PROTEIN 47B (RBP47B), is a sensor of PAs in Arabidopsis. PAs, including salicylic acid, 4-hydroxybenzoic acid, protocatechuic acid and so on, directly bind RBP47B, promote its phase separation and trigger SG formation accompanied by global translation inhibition. Salicylic acid-induced global translation inhibition depends on RBP47 family members. RBP47s regulate the proteome rather than the absolute quantity of SG. The rbp47 quadruple mutant shows a reduced sensitivity to the inhibitory effect of the PA mixture as well as to that of PA-rich rice when tested in a co-culturing ecosystem. In this Article, we identified the long sought-after PA sensor as RBP47B and illustrated that PA-induced SG-mediated translational inhibition was one of the PA perception mechanisms.

Aptamer-based nanointerferometer enables amplification-free ultrasensitive detection and differentiation of SARS-CoV-2 variants.

The state-of-the-art SARS-CoV-2 detection methods include qRT-PCR and antibody-based lateral flow assay (LFA) point-of-care tests. Despite the high sensitivity and selectivity, qRT-PCR is slow, expensive and needs well-trained operators. On the other extreme, LFA suffers from low sensitivity albeit its fast detection speed, low detection cost and ease of use. Therefore, the continuing COVID-19 pandemic calls for a SARS-CoV-2 detection method that is rapid, convenient and cost-effective without compromise in sensitivity. Here we provide a proof-of-principle demonstration of an optimized aptamer-based nanointerferometer that enables rapid and amplification-free detection of SARS-CoV-2 spike protein-coated pseudovirus directly from human saliva with the limit of detection (LOD) of about 400 copies per mL. This LOD is on par with that of qRT-PCR, making it 1000 to 100,000-fold more sensitive than commercial LFA tests. Using various combinations of negative selections during the screens for the aptamer targeting the receptor binding domain of the spike protein of SARS-CoV-2, we isolated two aptamers that can distinguish the Omicron and Delta variants. Integrating these two aptamers with LFA strips or the nanointerferometer sensors allows both detection and differentiation of the Omicron and Delta variants which has the potential to realize rapid triage of patients infected different SARS-CoV-2 variants.

Development of a structure-switching aptamer-based nanosensor for salicylic acid detection.

Salicylic acid (SA) is a phytohormone regulating immune responses against pathogens. SA and its derivatives can be found in diverse food products, medicines, cosmetics and preservatives. While salicylates have potential disease-preventative activity, they can also cause health problems to people who are hypersensitive. The current SA detection methods are costly, labor-intensive and require bulky instruments. In this study, a structure-switching aptamer-based nanopore thin film sensor was developed for cost-effective, rapid, sensitive and simple detection of SA in both buffer and plant extracts. SA is a challenging target for aptamer selection using conventional systemic evolution of ligands by exponential enrichment (SELEX) due to its small size and scarcity of reactive groups for immobilization. By immobilizing the SELEX library instead of SA and screening the library using a structure-switching SELEX approach, a high affinity SA aptamer was identified. The nanopore thin film sensor platform can detect as low as 0.1 µM SA. This is much better than the sensitivity of antibody-based detection method. This nanosensor also exhibited good selectivity among SA and its common metabolites and can detect SA in Arabidopsis and rice using only about 1 µl plant extracts within less than 30 min. The integration of SA aptamer and nanopore thin film sensor provides a promising solution for low-cost, rapid, sensitive on-site detection of SA.

An aptamer nanopore-enabled microsensor for detection of theophylline.

This paper reports an aptamer-based nanopore thin film sensor for detecting theophylline in the buffer solution and complex fluids including plant extracts and serum samples. Compared to antibody-based detection, aptamer-based detection offers many advantages such as low cost and high stability at elevated temperatures. Experiments found that this type of sensor can readily detect theophylline at a concentration as low as 0.05µM, which is much lower than the detection limit of current lab-based equipment such as liquid chromatography (LC). Experiments also found that the aptamer-based sensor has good specificity, selectivity, and reasonable reusability with a significantly improved dynamic detection range. By using the same nanopore thin film sensors as the reference sensors to further mitigate the non-specific binding effect, the theophylline in plant extracts and serum has been detected. Only a small amount (~1µL) of plant extracts or serum samples is required to measure theophylline. Its low cost and ease-of-operation make this type of sensor suitable for point-of-care application to monitor the theophylline level of patients in real time.

A genome-wide analysis of the expansin genes in Malus × Domestica.

Expansins were first identified as cell wall-loosening proteins; they are involved in regulating cell expansion, fruits softening and many other physiological processes. However, our knowledge about the expansin family members and their evolutionary relationships in fruit trees, such as apple, is limited. In this study, we identified 41 members of the expansin gene family in the genome of apple (Malus × Domestica L. Borkh). Phylogenetic analysis revealed that expansin genes in apple could be divided into four subfamilies according to their gene structures and protein motifs. By phylogenetic analysis of the expansins in five plants (Arabidopsis, rice, poplar, grape and apple), the expansins were divided into 17 subgroups. Our gene duplication analysis revealed that whole-genome and chromosomal-segment duplications contributed to the expansion of Mdexpansins. The microarray and expressed sequence tag (EST) data showed that 34 Mdexpansin genes could be divided into five groups by the EST analysis; they may also play different roles during fruit development. An expression model for MdEXPA16 and MdEXPA20 showed their potential role in developing fruit. Overall, our study provides useful data and novel insights into the functions and regulatory mechanisms of the expansin genes in apple, as well as their evolution and divergence. As the first step towards genome-wide analysis of the expansin genes in apple, our results have established a solid foundation for future studies on the function of the expansin genes in fruit development.

Arabidopsis SAG protein containing the MDN1 domain participates in seed germination and seedling development by negatively regulating ABI3 and ABI5.

Three proteins containing a midasin homologue 1 (MDN1) domain from the yeast Solanum chacoense and Arabidopsis thaliana have important functions in yeast survival, seed development, and female gametogenesis. In this study, a novel protein containing the MDN1 domain from Arabidopsis negatively regulated abscisic acid (ABA) signalling during seed germination. Seeds of a T-DNA insertion line of this gene exhibited increased sensitivity to ABA during seed germination and seedling development (named sag). By contrast, seeds with overexpressed AtSAG (OX2) were less sensitive to ABA. The seeds of the sag mutant showed similar sensitivity to high concentrations of mannitol and NaCl during these stages. AtSAG was also highly expressed in germinating seeds. However, ABA-induced AtSAG expression remained almost unchanged. ABA-responsive marker genes, including ABI3, ABI5, Em1, Em6, RD29A, and RAB18, were upregulated in sag mutants but were downregulated in OX2. Genetic analyses indicated that the function of AtSAG in ABA signalling depended on ABI3 and ABI5. The expression of some target genes of ABI3 and ABI5, such as seed storage protein and oleosin genes, was induced higher by ABA in sag mutants than in wild-type germinated seeds, even higher than in abi5 mutants. This finding indicated that other regulators similar to ABI3 or ABI5 played a role during these stages. Taken together, these results indicate that AtSAG is an important negative regulator of ABA signalling during seed germination and seedling development.

Genome-wide analysis and identification of stress-responsive genes of the NAM-ATAF1,2-CUC2 transcription factor family in apple.

NAC (NAM, ATAF1,2, and CUC2) proteins constitute one of the largest families of plant-specific transcription factors. To date, little is known about the NAC genes in the apple (Malus domestica). In this study, a total of 180 NAC genes were identified in the apple genome and were phylogenetically clustered into six groups (I-VI) with the NAC genes from Arabidopsis and rice. The predicted apple NAC genes were distributed across all of 17 chromosomes at various densities. Additionally, the gene structure and motif compositions of the apple NAC genes were analyzed. Moreover, the expression of 29 selected apple NAC genes was analyzed in different tissues and under different abiotic stress conditions. All of the selected genes, with the exception of four genes, were expressed in at least one of the tissues tested, which indicates that the NAC genes are involved in various aspects of the physiological and developmental processes of the apple. Encouragingly, 17 of the selected genes were found to respond to one or more of the abiotic stress treatments, and these 17 genes included not only the expected 7 genes that were clustered with the well-known stress-related marker genes in group IV but also 10 genes located in other subgroups, none of which contains members that have been reported to be stress-related. To the best of our knowledge, this report describes the first genome-wide analysis of the apple NAC gene family, and the results should provide valuable information for understanding the classification and putative functions of this family.

A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana.

The molecular mechanism for sensing and transducing the stress signals initiated by K(+) deprivation in plants remains unknown. Here, we found that the expression of AtHELPS, an Arabidopsis DExD/H box RNA helicase gene, was induced by low-K(+), zeatin and cold treatments, and downregulated by high-K(+) stress. To further investigate the expression pattern of AtHELPS, pAtHELPS::GUS transgenic plants were generated. Histochemical staining indicated that AtHELPS is mainly expressed in the young seedlings and vascular tissues of leaves and roots. Using both helps mutants and overexpression lines, we observed that, in the low-K(+) condition, AtHELPS affected Arabidopsis seed germination and plant weight. Interestingly, the mRNA levels of AKT1, CBL1/9 and CIPK23 in the helps mutants were much higher than in the overexpression lines under low-K(+) stress. Moreover, under low-K(+) stress, the helps mutants displayed increased K(+) influx, whereas the overexpression line of AtHELPS had a lower flux rate in the roots by the noninvasive micro-test technique. Taken together, these results provide information for the functional analysis of plant DEVH box RNA helicases, and suggest that AtHELPS, as an important negative regulator, plays a role in K(+) deprivation stress.