A role of age-dependent DNA methylation reprogramming in regulating the regeneration capacity of Boea hygrometrica leaves.

Plants can regenerate new individuals under appropriate culture conditions. Although the molecular basis of shoot regeneration has steadily been unraveled, the role of age-dependent DNA methylation status in the regulation of explant regeneration remains practically unknown. Here, we established an effective auxin/cytokinin-induced shoot regeneration system for the resurrection plant Boea hygrometrica via direct organogenesis and observed that regeneration was postponed with increasing age of donor plants. Global transcriptome analysis revealed significant upregulation of genes required for hormone signaling and phenylpropanoid biosynthesis and downregulation of photosynthetic genes during regeneration. Transcriptional changes in the positive/negative regulators and cell wall-related proteins involved in plant regeneration, such as ELONGATED HYPOCOTYL5 (HY5), LATERAL ORGAN BOUNDARIES DOMAIN, SHOOT-MERISTEMLESS, and WUSCHEL, were associated with the regeneration process. Comparison of DNA methylation profiling between leaves from young seedlings (YL) and mature plants (ML) revealed increased asymmetrical methylation in ML, which was predominantly distributed in promoter regions of genes, such as HY5 and a member of ABA-responsive element (ABRE) binding protein/ABRE binding factor, as well as genes encoding glycine-rich cell wall structural protein, CENTRORADIALIS-like protein, and beta-glucosidase 40-like essential for shoot meristem and cell wall architecture. Their opposite transcription response in ML explants during regeneration compared with those from YL demonstrated the putative involvement of DNA methylation in regeneration. Moreover, a significant lower expression of DNA glycosylase-lyase required for DNA demethylation in ML was coincident with its postponed regeneration compared with those in YL. Taken together, our results suggest a role of promoter demethylation in B. hygrometrica regeneration.

Weighted Gene Co-expression Network Analysis (WGCNA) Reveals the Hub Role of Protein Ubiquitination in the Acquisition of Desiccation Tolerance in Boea hygrometrica.

Boea hygrometrica can survive extreme drought conditions and has been used as a model to study desiccation tolerance. A genome-wide transcriptome analysis of B. hygrometrica showed that the plant can survive rapid air-drying after experiencing a slow soil-drying acclimation phase. In addition, a weighted gene co-expression network analysis was used to study the transcriptomic datasets. A network comprising 22 modules was constructed, and seven modules were found to be significantly related to desiccation response using an enrichment analysis. Protein ubiquitination was observed to be a common process linked to hub genes in all the seven modules. Ubiquitin-modified proteins with diversified functions were identified using immunoprecipitation coupled with mass spectrometry. The lowest level of ubiquitination was noted at the full soil drying priming stage, which coincided the accumulation of dehydration-responsive gene BhLEA2. The highly conserved RY motif (CATGCA) was identified from the promoters of ubiquitin-related genes that were downregulated in the desiccated samples. An in silico gene expression analysis showed that the negative regulation of ubiquitin-related genes is potentially mediated via a B3 domain-containing transcription repressor VAL1. This study suggests that priming may involve the transcriptional regulation of several major processes, and the transcriptional regulation of genes in protein ubiquitination may play a hub role to deliver acclimation signals to posttranslational level in the acquisition of desiccation tolerance in B. hygrometrica.

The resurrection genome of Boea hygrometrica: A blueprint for survival of dehydration.

"Drying without dying" is an essential trait in land plant evolution. Unraveling how a unique group of angiosperms, the Resurrection Plants, survive desiccation of their leaves and roots has been hampered by the lack of a foundational genome perspective. Here we report the ∼1,691-Mb sequenced genome of Boea hygrometrica, an important resurrection plant model. The sequence revealed evidence for two historical genome-wide duplication events, a compliment of 49,374 protein-coding genes, 29.15% of which are unique (orphan) to Boea and 20% of which (9,888) significantly respond to desiccation at the transcript level. Expansion of early light-inducible protein (ELIP) and 5S rRNA genes highlights the importance of the protection of the photosynthetic apparatus during drying and the rapid resumption of protein synthesis in the resurrection capability of Boea. Transcriptome analysis reveals extensive alternative splicing of transcripts and a focus on cellular protection strategies. The lack of desiccation tolerance-specific genome organizational features suggests the resurrection phenotype evolved mainly by an alteration in the control of dehydration response genes.

Efficient modulation of photosynthetic apparatus confers desiccation tolerance in the resurrection plant Boea hygrometrica.

Boea hygrometrica (B. hygrometrica) can tolerate severe desiccation and resume photosynthetic activity rapidly upon water availability. However, little is known about the mechanisms by which B. hygrometrica adapts to dehydration and resumes competence upon rehydration. Here we determine how B. hygrometrica deals with oxidative stress, excessive excitation/electron pressures as well as photosynthetic apparatus modulation during dehydration/rehydration. By measuring ROS generation and scavenging efficiency, we found that B. hygrometrica possesses efficient strategies to maintain cellular redox homeostasis. Transmission electron microscopy (TEM) analysis revealed a remarkable alteration of chloroplast architecture and plastoglobules (PGs) accumulation during dehydration/rehydration. Pulse-amplitude modulated (PAM) chlorophyll fluorescence measurements, P700 redox assay as well as chlorophyll fluorescence emission spectra analysis on leaves of B. hygrometrica during dehydration/rehydration were also performed. Results showed that the photochemical activity of PSII as well as photoprotective energy dissipation in PSII undergo gradual inactivation/activation during dehydration/rehydration in B. hygrometrica; PSI activity is relatively induced upon water deficit, and dehydration leads to physical interaction between PSI and LHCII. Furthermore, blue-native polyacrylamide gel electrophoresis (BN-PAGE) and immunoblot analysis revealed that the protein abundance of light harvesting complexes decrease markedly along with internal water deficit to restrict light absorption and attenuate electron transfer, resulting in limited light excitation and repressed photosynthesis. In contrast, many thylakoid proteins remain at a basal level even after full dehydration. Taken together, our study demonstrated that efficient modulation of cellular redox homeostasis and photosynthetic activity confers desiccation tolerance in B. hygrometrica.

Global Transcriptome Analysis Reveals Acclimation-Primed Processes Involved in the Acquisition of Desiccation Tolerance in Boea hygrometrica.

Boea hygrometrica resurrection plants require a period of acclimation by slow soil-drying in order to survive a subsequent period of rapid desiccation. The molecular basis of this observation was investigated by comparing gene expression profiles under different degrees of water deprivation. Transcripts were clustered according to the expression profiles in plants that were air-dried (rapid desiccation), soil-dried (gradual desiccation), rehydrated (acclimated) and air-dried after acclimation. Although phenotypically indistinguishable, it was shown by principal component analysis that the gene expression profiles in rehydrated, acclimated plants resemble those of desiccated plants more closely than those of hydrated acclimated plants. Enrichment analysis based on gene ontology was performed to deconvolute the processes that accompanied desiccation tolerance. Transcripts associated with autophagy and α-tocopherol accumulation were found to be activated in both air-dried, acclimated plants and soil-dried non-acclimated plants. Furthermore, transcripts associated with biosynthesis of ascorbic acid, cell wall catabolism, chaperone-assisted protein folding, respiration and macromolecule catabolism were activated and maintained during soil-drying and rehydration. Based on these findings, we hypothesize that activation of these processes leads to the establishment of an optimal physiological and cellular state that enables tolerance during rapid air-drying. Our study provides a novel insight into the transcriptional regulation of critical priming responses to enable survival following rapid dehydration in B. hygrometrica.

Common and Specific Mechanisms of Desiccation Tolerance in Two Gesneriaceae Resurrection Plants. Multiomics Evidences.

Environmental stress, especially water deficiency, seriously limits plant distribution and crop production worldwide. A small group of vascular angiosperm plants termed "resurrection plants," possess desiccation tolerance (DT) to withstand dehydration and to recover fully upon rehydration. In recent years, with the rapid development of life science in plants different omics technologies have been widely applied in resurrection plants to study DT. Boea hygrometrica is native in East and Southeast Asia, and Haberlea rhodopensis is endemic to the Balkans in Europe. They are both resurrection pants from Gesneriaceae family. This paper reviews recent advances in transcriptome and metabolome, and discusses the differences and similarities of DT features between both species. Finally, we believe we provide novel insights into understanding the mechanisms underlying the acquisition and evolution of desiccation tolerance of the resurrection plants that could substantially contribute to develop new approaches for agriculture to overcome water deficiency in future.

BhbZIP60 from Resurrection Plant Boea hygrometrica Is an mRNA Splicing-Activated Endoplasmic Reticulum Stress Regulator Involved in Drought Tolerance.

Adverse environmental conditions cause endoplasmic reticulum (ER) stress in plants. To mitigate ER stress damage, ER associated transcription factors and inositol-requiring enzyme-1 (IRE1)-mediated bZIP60 mRNA splicing are activated in plants. A drought-induced gene, encoding the ortholog of AtbZIP60, was identified in the resurrection plant Boea hygrometrica, termed BhbZIP60. In response to ER stress and dehydration, BhbZIP60 mRNA can be spliced to create a frame shift in the C terminus by the excision of 23b segment in a manner of its ortholog in other plants, thus translocating to the nucleus instead of the cytoplasm. The splicing-activated BhbZIP60 (BhbZIP60S) could function in the same way as its Arabidopsis ortholog by restoring the molecular phenotype of the mutant atbzip60. When overexpressed in Arabidopsis, BhbZIP60S provided transgenic plants with enhanced tolerance to drought, tunicamycin and mannitol stresses with upregulation of the expressions of ER quality control (QC) genes (BiP2, BiP3, CNX1, and sPDI) and abscisic acid (ABA) responsive genes (RD29A, RAB18, and RD17). Furthermore, in the yeast one-hybrid system, BhbZIP60S was capable of interacting with ER stress responsive elements (ERSE and ERSE-II) that exist in the promoters of known ER-QC genes, but not binding to ABA responsive cis-elements (ABREs). Our results demonstrated that drought-induced BhbZIP60 may have a function in drought tolerance via the splicing-activated BhbZIP60S to mediate ER-QC by direct binding to the promoters of ER-QC genes. This study evidently demonstrates the involvement of ER-QC in the drought tolerance of Arabidopsis and the desiccation tolerance of the resurrection plant B. hygrometrica.

A comparative study on Ca content and distribution in two Gesneriaceae species reveals distinctive mechanisms to cope with high rhizospheric soluble calcium.

Excessive Ca is toxic to plants thus significantly affects plant growth and species distribution in Ca-rich karst areas. To understand how plants survive high Ca soil, laboratory experiments were established to compare the physiological responses and internal Ca distribution in organ, tissue, cell, and intracellular levels under different Ca levels for Lysionotus pauciflorus and Boea hygrometrica, two karst habitant Gesneriaceae species in Southwest China. In the controlled condition, L. pauciflorus could survive as high as 200 mM rhizospheric soluble Ca, attributed to a series of physiological responses and preferential storage that limited Ca accumulation in chloroplasts of palisade cells. In contrast, B. hygrometrica could survive only 20 mM rhizospheric soluble Ca, but accumulated a high level of internal Ca in both palisade and spongy cells without disturbance on photosynthetic activity. By phenotype screening of transgenic plants expressing high Ca-inducible genes from B. hygrometrica, the expression of BhDNAJC2 in A. thaliana was found to enhance plant growth and photosynthesis under high soluble Ca stress. BhDNAJC2 encodes a recently reported heat shock protein (HSP) 40 family DnaJ-domain protein. The Ca-resistant phenotype of BhDNAJC2 highlights the important role of chaperone-mediated protein quality control in Ca tolerance in B. hygrometrica. Taken together, our results revealed that distinctive mechanisms were employed in the two Gesneriaceae karst habitants to cope with a high Ca environment.

Understanding desiccation tolerance using the resurrection plant Boea hygrometrica as a model system.

Vegetative tissues of Boea hygrometrica, a member of the Gesneriaceae family, can tolerate severe water loss to desiccated state and fully recover upon rehydration. Unlike many other so called "resurrection plants," the detached leaves of B. hygrometrica also possess the same level of capacity for desiccation tolerance (DT) as that of whole plant. B. hygrometrica is distributed widely from the tropics to northern temperate regions in East Asia and grows vigorously in areas around limestone rocks, where dehydration occurs frequently, rapidly, and profoundly. The properties of detached B. hygrometrica leaves and relative ease of culture have made it a useful system to study the adaptive mechanisms of DT. Extensive studies have been conducted to identify the physiological, cellular, and molecular mechanisms underlying DT in the last decade, including specific responses to water stress, such as cell wall folding and pigment-protein complex stabilizing in desiccated leaves. In this review, the insight into the structural, physiological, and biochemical, and molecular alterations that accompany the acquisition of DT in B. hygrometrica is described. Finally a future perspective is proposed, with an emphasis on the emerging regulatory roles of retroelements and histone modifications in the acquisition of DT, and the need of establishment of genome sequence database and high throughput techniques to identify novel regulators for fully understanding of the matrix of DT.