Dominant-negative chaperonin mutation ptCPN60α1S57F uncovers redundancy in chloroplast rRNA processing.

The biogenesis of functional forms of chloroplast ribosomal RNAs (rRNAs) is crucial for the translation of chloroplast mRNAs into polypeptides. However, the molecular mechanisms underlying the proper processing and maturation of chloroplast rRNA species are poorly understood. Through a genetic approach, we isolated and characterized an Arabidopsis mutant, α1-4, harboring a missense mutation in the plastid chaperonin-60α1 gene. Using allelism tests and transgenic manipulation, we determined functional redundancy among ptCPN60 subunits. The ptCPN60α1S57F mutation caused specific defects in the formation of chloroplast rRNA species, including 23S, 5S, and 4.5S rRNAs, but not 16S rRNAs. Allelism tests suggested that the dysfunctional ptCPN60α1S57F competes with other members of the ptCPN60 family. Indeed, overexpression of the ptCPN60α1S57F protein in wild-type plants mimicked the phenotypes observed in the α1-4 mutant, while increasing the endogenous transcriptional levels of ptCPN60α2, ß1, ß2, and ß3 in the α1-4 mutant partially mitigated the abnormal fragmentation processing of chloroplast 23S, 5S, and 4.5S rRNAs. Furthermore, we demonstrated functional redundancy between ptCPN60ß1 and ptCPN60ß2 in chloroplast rRNA processing through double-mutant analysis. Collectively, our data reveal a novel physiological role of ptCPN60 subunits in generating the functional rRNA species of the large 50S ribosomal subunit in Arabidopsis chloroplasts.

Ethylene is the key phytohormone to enhance arsenic resistance in Arabidopsis thaliana.

The toxic metalloid arsenic is prevalent in the environment and poses a threat to nearly all organisms. However, the mechanism by which phytohormones modulate arsenic resistance is not well-understood. Therefore, we analyzed multiple phytohormones based on the results of transcriptome sequencing, content changes, and related mutant growth under arsenic stress. We found that ethylene was the key phytohormone in Arabidopsis thaliana response to arsenic. Further investigation showed the ethylene-overproducing mutant eto1-1 generated less malondialdehyde (MDA), H2O2, and O2•- under arsenic stress compared to wild-type, while the ethylene-insensitive mutant ein2-5 displayed opposite patterns. Compared to wild-type, eto1-1 accumulated a smaller amount of arsenic and a larger amount of non-protein thiols. Additionally, the immediate ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), enhanced resistance to arsenic in wide-type, but not in mutants with impaired detoxification capability (i.e., cad1-3, pad2-1, abcc1abcc2), which confirmed that ethylene regulated arsenic detoxification by enhancing arsenic chelation. ACC also upregulated the expression of gene(s) involved in arsenic detoxification, among which ABCC2 was directly transcriptionally activated by the ethylene master transcription factor ethylene-insensitive 3 (EIN3). Overall, our study shows that ethylene is the key phytohormone to enhance arsenic resistance by reducing arsenic accumulation and promoting arsenic detoxification at both physiological and molecular levels.

OsGLP8-7 interacts with OsPRX111 to detoxify excess copper in rice.

Lignin is a phenolic biopolymer generated from phenylpropanoid pathway in the secondary cell wall and is required for defense of plants against various stress. Although the fact of stress-induced lignin deposition has been clearly demonstrated, it remains largely elusive how the formation of lignin is promoted under Cu stress. The present study showed that OsGLP8-7, an extracellular glycoprotein of rice (Oryza sativa L.), plays an important function against Cu stress. The loss function of OsGLP8-7 results in Cu sensitivity whereas overexpression of OsGLP8-7 scavenges Cu-induced superoxide anion (O2•-). OsGLP8-7 interacts with apoplastic peroxidase111 (OsPRX111) and elevates OsPRX111 stability when exposed to excess Cu. In OsGLP8-7 overexpressing (OE) lines, the retention of Cu within cell wall limiting Cu uptake into cytoplasm is attributed to the enhanced lignification required for Cu tolerance. Exogenous application of a lignin inhibitor can impair the Cu tolerance of transgenic Arabidopsis lines overexpressing OsGLP8-7. In addition, co-expression of OsGLP8-7 and OsPRX111 genes in tobacco leaves leads to an improved lignin deposition compared to leaves expressing each gene individually or the empty vector. Taken together, our findings provided the convincing evidences that the interaction between OsGLP8-7 and OsPRX111 facilitates effectively lignin polymerization, thereby contributing to Cu tolerance in rice.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

Salt-induced inhibition of rice seminal root growth is mediated by ethylene-jasmonate interaction.

The phytohormones ethylene and jasmonate play important roles in the adaptation of rice plants to salt stress. However, the molecular interactions between ethylene and jasmonate on rice seminal root growth under salt stress are unknown. In this study, the effects of NaCl on the homeostasis of ethylene and jasmonate, and on rice seminal root growth were investigated. Our results indicate that NaCl treatment promotes ethylene biosynthesis by up-regulating the expression of ethylene biosynthesis genes, whereas NaCl-induced ethylene does not inhibit rice seminal root growth directly, but rather indirectly, by promoting jasmonate biosynthesis. NaCl treatment also promotes jasmonate biosynthesis through an ethylene-independent pathway. Moreover, NaCl-induced jasmonate reduces meristem cell number and cell division activity via down-regulated expression of Oryza sativa PLETHORA (OsPLT) and cell division-related genes, respectively. Additionally, NaCl-induced jasmonate inhibits seminal root cell elongation by down-regulating the expression of cell elongation-related genes. Overall, salt stress promotes jasmonate biosynthesis through ethylene-dependent and -independent pathways in rice seminal roots, and jasmonate inhibits rice seminal root growth by inhibiting root meristem cell proliferation and root cell elongation.

Promotion of growth and phytoextraction of cadmium and lead in Solanum nigrum L. mediated by plant-growth-promoting rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR) are a specific category of microbes that improve plant growth and promote greater tolerance to metal stress through their interactions with plant roots. We evaluated the effects of phytoremediation combining the cadmium accumulator Solanum nigrum L. and two Cd- and Pb-resistant bacteria isolates. To understand the interaction between PGPR and their host plant, we conducted greenhouse experiments with inoculation treatments at Nanjing Agricultural University (Jiangsu Province, China), in June 2018. Two Cd- and Pb-resistant PGPR with various growth-promoting properties were isolated from heavy metal-contaminated soil. 16S rRNA analyses indicated that the two isolates were Bacillus genus, and they were named QX8 and QX13. Pot experiments demonstrated that inoculation may improve the rhizosphere soil environment and promote absorption of Fe and P by plants. Inoculation with QX8 and QX13 also enhanced the dry weight of shoots (1.36- and 1.7-fold, respectively) and roots (1.42- and 1.96-fold) of plants growing in Cd- and Pb-contaminated soil, and significantly increased total Cd (1.28-1.81 fold) and Pb (1.08-1.55 fold) content in aerial organs, compared to non-inoculated controls. We also detected increases of 23% and 22% in the acid phosphatase activity of rhizosphere soils inoculated with QX8 and QX13, respectively. However, we did not detect significant differences between inoculated and non-inoculated treatments in Cd and Pb concentrations in plants and available Cd and Pb content in rhizosphere soils. We demonstrated that PGPR-assisted phytoremediation is a promising technique for remediating heavy metal-contaminated soils, with the potential to enhance phytoremediation efficiency and improve soil quality.

The root iron transporter 1 governs cadmium uptake in Vicia sativa roots.

Cadmium is a non-essential element for plants and that inhibits plant growth and development. The Zhangye Mawan (ZM) variety of Vicia sativa is more sensitive to Cd toxicity than that Lanjian 3# (L3) variety, but the underlying mechanism is not fully understood. Here, we demonstrated that ZM showed higher Cd accumulation than L3 based on root Cd content and Cd fluorescence intensity in root protoplasts. VsRIT1, a member of the ZIP (ZRT/IRT-like protein) family, showed expression levels in ZM roots 8-fold higher than those in L3 roots under Cd exposure. VsRIT1 expression increased Cd transport and accumulation in Arabidopsis and yeast. These suggests that VsRIT1 participates in Cd uptake by V. sativa roots. Furthermore, ZM root tips have a higher capacity for transient Cd influx than L3 roots when exposed to Cd alone or Cd and iron (Fe) together, owing to the higher VsRIT1 expression in ZM. Our findings also imply that Cd may compete with Fe or/and zinc (Zn) for uptake via VsRIT1 in V. sativa or yeast.

Differential effects of three amendments on the immobilisation of cadmium and lead for Triticum aestivum grown on polluted soil.

Conventional chemical soil amendments and novel material biochars have been widely reported for the immobilisation of cadmium (Cd) and lead (Pb) in polluted soil. However, information regarding their comparative effectiveness is poor. In the present study, rice husk biochar (RHB) was compared with two chemical soil amendments including hydroxyapatite (HAP) and hydrated lime (HDL) for their effectiveness to enhance plant growth and the reduction of Cd uptake and translocation by Triticum aestivum L. grown in heavy-metal-polluted soil. Compared with control and two chemical soil amendments, RHB rapidly improved wheat growth. The HAP, HDL, and RHB treated plants retained Cd and Pb in roots and restricted their translocation. The RHB treatment had the best effect on growth, yield promotion and the reduction of Cd and Pb in wheat grain. Furthermore, the soils treated with RHB and HAP showed lower DTPA-extracted Cd concentrations, and the maximum reduction was observed in HAP-amended soil. However, the DTPA-extracted Pb concentration was not significantly decreased after the application of two chemical soil amendments for 40 days; the maximum reduction was found in soil treated with RHB for 80 days. In all treatments, Cd in post-harvest soil was mainly present in exchangeable, carbonate bound, and Fe-Mn oxide Cd, while the dominant chemical form of Pb was Fe-Mn oxide Pb. Three soil amendments application decreased exchangeable and organic bound- Cd and Pb levels. HAP and RHB displayed significantly immobilisation for soil Cd and reduced translocation of heavy metal as well as its availability in soil, but the HAP had significant inhibition on growth of wheat in contaminated soil. Therefore, RHB shows a promising potential for the reduction of Cd and Pb bioaccumulation in grains from wheat grown on heavy-metal-polluted soils.

Comparative Transcriptome Analysis of Two Contrasting Soybean Varieties in Response to Aluminum Toxicity.

: Aluminum (Al) toxicity is a major factor limiting crop productivity on acid soils. Soybean (Glycine max) is an important oil crop and there is great variation in Al tolerance in soybean germplasms. However, only a few Al-tolerance genes have been reported in soybean. Therefore, the purpose of this study was to identify candidate Al tolerance genes by comparative transcriptome analysis of two contrasting soybean varieties in response to Al stress. Two soybean varieties, M90-24 (M) and Pella (P), which showed significant difference in Al tolerance, were used for RNA-seq analysis. We identified a total of 354 Al-tolerance related genes, which showed up-regulated expression by Al in the Al-tolerant soybean variety M and higher transcript levels in M than P under Al stress. These genes were enriched in the Gene Ontology (GO) terms of cellular glucan metabolic process and regulation of transcription. Five out of 11 genes in the enriched GO term of cellular glucan metabolic process encode cellulose synthases, and one cellulose synthase gene (Glyma.02G205800) was identified as the key hub gene by co-expression network analysis. Furthermore, treatment of soybean roots with a cellulose biosynthesis inhibitor decreased the Al tolerance, indicating an important role of cellulose production in soybean tolerance to Al toxicity. This study provides a list of candidate genes for further investigation on Al tolerance mechanisms in soybean.

Assembly and Annotation of a Draft Genome of the Medicinal Plant Polygonum cuspidatum.

Polygonum cuspidatum (Japanese knotweed, also known as Huzhang in Chinese), a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance.

Polyaspartate and liquid amino acid fertilizer are appropriate alternatives for promoting the phytoextraction of cadmium and lead in Solanum nigrum L.

Traditional metal chelators, such as ethylenediaminetetraacetic acid (EDTA), have been gradually replaced due to their poor biodegradability in soil and high risk of heavy metal leaching into groundwater, which pose high environmental risks to the health of humans and animals. In this study, a liquid amino acid fertilizer (LAAF, waste proteins from hydrolysates of animal carcasses) and polyaspartate (PASP) were used as additives to enhance the phytoextraction of cadmium (Cd) and lead (Pb) from contaminated soil. We conducted pot experiments to investigate the phytoextraction capacity of Solanum nigrum, a Cd accumulator, grown on soil highly contaminated with Cd and Pb in the absence (as controls) or presence of PASP and LAAF. Both PASP and LAAF significantly improved plant growth, Cd accumulation, and total Cd and Pb content in S. nigrum shoots and roots. PASP and LAAF application promoted Cd translocation from roots to shoots in S. nigrum and Cd bio-accessibility in rhizosphere soils, but this was not the case for Pb. Both PASP and LAAF increased Cd and Pb phytoextraction by S. nigrum plants, and Cd phytoextraction was more effective in LAAF-assisted S. nigrum than in PASP-assisted S. nigrum. These findings demonstrate that the low cost and ecofriendly features of recycled waste proteins make them good candidates for chelant-enhanced phytoextraction from heavy metal-contaminated soils.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

eIF2α kinases PERK and GCN2 act on FOXO to potentiate FOXO activity.

PERK and GCN2 are eIF2α kinases known to mediate the effects of ER stress and respond to an array of diverse stress stimuli. Previously, we reported that ER stress potentiates insulin resistance through PERK-mediated FOXO phosphorylation. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. Here we provide further evidence that FOXO is required for the functional output of PERK by showing that lowering FOXO activity ameliorates a PERK gain-of-function phenotype in Drosophila. More importantly, we present results demonstrating that GCN2 acts similarly to PERK to promote FOXO activity. Regulation of FOXO by GCN2 is evolutionarily conserved and can be compensated for by PERK. The combination of these mechanisms may contribute to the complex regulatory network between PERK, GCN2, and FOXO, which has been implicated in the development and progression of a variety of diseases.

Overexpression of a Functional Vicia sativa PCS1 Homolog Increases Cadmium Tolerance and Phytochelatins Synthesis in Arabidopsis.

Phytochelatins (PCs) catalyzed by phytochelatin synthases (PCS) are important for the detoxification of metals in plants and other living organisms. In this study, we isolated a PCS gene (VsPCS1) from Vicia sativa and investigated its role in regulating cadmium (Cd) tolerance. Expression of VsPCS1 was induced in roots of V. sativa under Cd stress. Analysis of subcellular localization showed that VsPCS1 was localized in the cytoplasm of mesophyll protoplasts of V. sativa. Overexpression of VsPCS1 (35S::VsPCS1, in wild-type background) in Arabidopsis thaliana could complement the defects of Cd tolerance of AtPCS1-deficent mutant (atpcs1). Compared with atpcs1 mutants, 35S::VsPCS1/atpcs1 (in AtPCS1-deficent mutant background) transgenic plants significantly lowered Cd-fluorescence intensity in mesophyll cytoplasm, accompanied with enhanced Cd-fluorescence intensity in the vacuoles, demonstrating that the increased Cd tolerance may be attributed to the increased PC-based sequestration of Cd into the vacuole. Furthermore, overexpressing VsPCS1 could enhance the Cd tolerance in 35S::VsPCS1, but have no effect on Cd accumulation and distribution, showing the same level of Cd-fluorescence intensity between 35S::VsPCS1 and wild-type (WT) plants. Further analysis indicated this increased tolerance in 35S::VsPCS1 was possibly due to the increased PCs-chelated Cd in cytosol. Taken together, a functional PCS1 homolog from V. sativa was identified, which hold a strong catalyzed property for the synthesis of high-order PCs that retained Cd in the cytosol rather the vacuole. These findings enrich the original model of Cd detoxification mediated by PCS in higher plants.