Structure and dynamics of microbial communities associated with the resurrection plant Boea hygrometrica in response to drought stress.

MAIN CONCLUSION: The resurrection plant Boea hygrometrica selectively recruits and assembles drought-specific microbial communities across the plant-soil compartments, which may benefit plant growth and fitness under extreme drought conditions. Plant-associated microbes are essential for facilitating plant growth and fitness under drought stress. The resurrection plant Boea hygrometrica in natural habitats with seasonal rainfall can survive rapid desiccation, yet their interaction with microbiomes under drought conditions remains unexplored. This study examined the bacterial and fungal microbiome structure and drought response across plant-soil compartments of B. hygrometrica by high-throughput amplicon sequencing of 16S rRNA gene and internal transcribed spacer. Our results demonstrated that the diversity, composition, and functional profile of the microbial community varied considerably across the plant-soil compartments and were strongly affected by drought stress. Bacterial and fungal diversity was significantly reduced from soil to endosphere and belowground to aboveground compartments. The compartment-specific enrichment of the dominant bacteria phylum Cyanobacteriota and genus Methylorubrum in leaf endosphere, genera Pseudonocardia in rhizosphere soil and Actinoplanes in root endosphere, and fungal phylum Ascomycota in the aboveground compartments and genera Knufia in root endosphere and Cladosporium in leaf endosphere composed part of the core microbiota with corresponding enrichment of beneficial functions for plant growth and fitness. Moreover, the recruitment of dominant microbial genera Sphingosinicella and Plectosphaerella, Ceratobasidiaceae mycorrhizal fungi, and numerous plant growth-promoting bacteria involving nutrient supply and auxin regulation was observed in desiccated B. hygrometrica plants. Our results suggest that the stable assembled drought-specific microbial community of B. hygrometrica may contribute to plant survival under extreme environments and provide valuable microbial resources for the microbe-mediated drought tolerance enhancement in crops.

DNA methylation-mediated modulation of rapid desiccation tolerance acquisition and dehydration stress memory in the resurrection plant Boea hygrometrica.

Pre-exposure of plants to various abiotic conditions confers improved tolerance to subsequent stress. Mild drought acclimation induces acquired rapid desiccation tolerance (RDT) in the resurrection plant Boea hygrometrica, but the mechanisms underlying the priming and memory processes remain unclear. In this study, we demonstrated that drought acclimation-induced RDT can be maintained for at least four weeks but was completely erased after 18 weeks based on a combination of the phenotypic and physiological parameters. Global transcriptome analysis identified several RDT-specific rapid dehydration-responsive genes related to cytokinin and phospholipid biosynthesis, nitrogen and carbon metabolism, and epidermal morphogenesis, most of which were pre-induced by drought acclimation. Comparison of whole-genome DNA methylation revealed dehydration stress-responsive hypomethylation in the CG, CHG, and CHH contexts and acclimation-induced hypermethylation in the CHH context of the B. hygrometrica genome, consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with gene expression levels in both acclimated and dehydrated plants but showed no association with transcriptional divergence during the procedure. Nevertheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation, and post-translational protein modification during acclimation, growth, and rapid dehydration stress response. It was also associated with the dehydration stress-induced upregulation of memory genes, including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like, and UDP-sugar pyrophosphorylase, which may contribute directly or indirectly to the improvement of dehydration tolerance in B. hygrometrica plants. Altogether, our findings demonstrate the potential implications of DNA methylation in dehydration stress memory and, therefore, provide a molecular basis for enhanced dehydration tolerance in plants induced by drought acclimation.

Modulation of volatile compound metabolome and transcriptome in grape berries exposed to sunlight under dry-hot climate.

BACKGROUND: Basal leaf removal is widely practiced to increase grape cluster sunlight exposure that controls berry rot and improves quality. Studies on its influence on volatile compounds in grape berries have been performed mostly in Mediterranean or marine climate regions. It is uncertain whether similar efficiency can be achieved when grape berries are grown under continental climate. This study aimed to dissect the variation in volatile compound production and transcriptome in sunlight-exposed grape berries in a dry-hot climate region and to propose the key genes related to the variation. RESULTS: Four cluster sunlight exposure strategies, including basal leaf removal at pepper-corn size stage, leaf removal at véraison (LR-V), leaf moving at véraison (LM-V), and half-leaf removal at véraison, were implemented at the north foot of the Mt. Tianshan region of northwestern China. Various cluster exposure treatments resulted in a decline in the concentrations of norisoprenoids and monoterpenes in ripening grape berries. Both ß-carotene and lutein, the substrates of norisoprenoid biosynthesis, were reduced by cluster sunlight exposure. K-means cluster analysis showed that some genes involved in biosynthesis such as VviTPS55, VviTPS60, VviTPS66, VviCCD4a and VviCCD4b exhibited lower expression levels in exposed berries at least at one of the tested stages. Two C6-derived esters with fruity attributes, ethyl hexanoate and hexyl acetate, were reduced markedly. In contrast, main C6 alcohol compound levels were elevated in the LR-V- and LM-V-treated grape berries, which corresponded to the up-regulated expression of VviLOXA, VviLOXO and VviADH1 in the oxylipin pathway. Most of the differentially expressed genes in the exposed and control berries were enriched to the "stress response" processes, and this transcriptome difference was accumulated as the berries matured. Besides, LR-V treatment stimulated a significant up-regulation in photosynthesis-related genes in the grape berries, which did not happen with LM-V treatment. CONCLUSIONS: Cluster sunlight exposure in dry-hot climate viticulture resulted in different volatile-targeted transcriptomic and metabolic responses from those obtained in the temperate Mediterranean or marine climate region. Therefore, a modified canopy management should be adopted to improve the aroma of grape berries.

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.

Comparative physiological, metabolomic, and transcriptomic analyses reveal developmental stage-dependent effects of cluster bagging on phenolic metabolism in Cabernet Sauvignon grape berries.

BACKGROUND: Light conditions significantly influence grape berry ripening and the accumulation of phenolic compounds, but the underlying molecular basis remains partially understood. Here, we applied integrated transcriptomics and pathway-level metabolomics analyses to investigate the effect of cluster bagging during various developmental stages on phenolic metabolism in Cabernet Sauvignon grapes. RESULTS: Bagging treatments had limited effects on berry quality attributes at harvest and did not consistently affect phenolic acid biosynthesis between seasons. Significantly elevated flavan-3-ol and flavonol contents were detected in re-exposed berries after bagging during early-developmental stages, while bagging after véraison markedly inhibited skin anthocyanin accumulation. Several anthocyanin derivatives and flavonol glycosides were identified as marker phenolic metabolites for distinguishing bagged and non-bagged grapes. Coordinated transcriptional changes in the light signaling components CRY2 and HY5/HYHs, transcription regulator MYBA1, and enzymes LAR, ANR, UFGT and FLS4, coincided well with light-responsive biosynthesis of the corresponding flavonoids. The activation of multiple hormone signaling pathways after both light exclusion and re-exposure treatments was inconsistent with the changes in phenolic accumulation, indicating a limited role of plant hormones in mediating light/darkness-regulated phenolic biosynthesis processes. Furthermore, gene-gene and gene-metabolite network analyses discovered that the light-responsive expression of genes encoding bHLH, MYB, WRKY, NAC, and MADS-box transcription factors, and proteins involved in genetic information processing and epigenetic regulation such as nucleosome assembly and histone acetylation, showed a high positive correlation with grape berry phenolic accumulation in response to different light regimes. CONCLUSIONS: Altogether, our findings provide novel insights into the understanding of berry phenolic biosynthesis under light/darkness and practical guidance for improving grape features.

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.

Insights into transcriptional characteristics and homoeolog expression bias of embryo and de-embryonated kernels in developing grain through RNA-Seq and Iso-Seq.

Bread wheat (Triticum aestivum L.) is an allohexaploid, and the transcriptional characteristics of the wheat embryo and endosperm during grain development remain unclear. To analyze the transcriptome, we performed isoform sequencing (Iso-Seq) for wheat grain and RNA sequencing (RNA-Seq) for the embryo and de-embryonated kernels. The differential regulation between the embryo and de-embryonated kernels was found to be greater than the difference between the two time points for each tissue. Exactly 2264 and 4790 tissue-specific genes were found at 14 days post-anthesis (DPA), while 5166 and 3784 genes were found at 25 DPA in the embryo and de-embryonated kernels, respectively. Genes expressed in the embryo were more likely to be related to nucleic acid and enzyme regulation. In de-embryonated kernels, genes were rich in substance metabolism and enzyme activity functions. Moreover, 4351, 4641, 4516, and 4453 genes with the A, B, and D homoeoloci were detected for each of the four tissues. Expression characteristics suggested that the D genome may be the largest contributor to the transcriptome in developing grain. Among these, 48, 66, and 38 silenced genes emerged in the A, B, and D genomes, respectively. Gene ontology analysis showed that silenced genes could be inclined to different functions in different genomes. Our study provided specific gene pools of the embryo and de-embryonated kernels and a homoeolog expression bias model on a large scale. This is helpful for providing new insights into the molecular physiology of wheat.

Transcriptome reprogramming during severe dehydration contributes to physiological and metabolic changes in the resurrection plant Haberlea rhodopensis.

BACKGROUND: Water shortage is a major factor that harms agriculture and ecosystems worldwide. Plants display various levels of tolerance to water deficit, but only resurrection plants can survive full desiccation of their vegetative tissues. Haberlea rhodopensis, an endemic plant of the Balkans, is one of the few resurrection plants found in Europe. We performed transcriptomic analyses of this species under slight, severe and full dehydration and recovery to investigate the dynamics of gene expression and associate them with existing physiological and metabolomics data. RESULTS: De novo assembly yielded a total of 142,479 unigenes with an average sequence length of 1034 nt. Among them, 18,110 unigenes were differentially expressed. Hierarchical clustering of all differentially expressed genes resulted in seven clusters of dynamic expression patterns. The most significant expression changes, involving more than 15,000 genes, started at severe dehydration (~ 20% relative water content) and were partially maintained at full desiccation (< 10% relative water content). More than a hundred pathways were enriched and functionally organized in a GO/pathway network at the severe dehydration stage. Transcriptomic changes in key pathways were analyzed and discussed in relation to metabolic processes, signal transduction, quality control of protein and DNA repair in this plant during dehydration and rehydration. CONCLUSION: Reprograming of the transcriptome occurs during severe dehydration, resulting in a profound alteration of metabolism toward alternative energy supply, hormone signal transduction, and prevention of DNA/protein damage under very low cellular water content, underlying the observed physiological and metabolic responses and the resurrection behavior of H. rhodopensis.

Light-induced Variation in Phenolic Compounds in Cabernet Sauvignon Grapes (Vitis vinifera L.) Involves Extensive Transcriptome Reprogramming of Biosynthetic Enzymes, Transcription Factors, and Phytohormonal Regulators.

Light environments have long been known to influence grape (Vitis vinifera L.) berry development and biosynthesis of phenolic compounds, and ultimately affect wine quality. Here, the accumulation and compositional changes of hydroxycinnamic acids (HCAs) and flavonoids, as well as global gene expression were analyzed in Cabernet Sauvignon grape berries under sunlight exposure treatments at different phenological stages. Sunlight exposure did not consistently affect the accumulation of berry skin flavan-3-ol or anthocyanin among different seasons due to climatic variations, but increased HCA content significantly at véraison and harvest, and enhanced flavonol accumulation dramatically with its timing and severity degree trend. As in sunlight exposed berries, a highly significant correlation was observed between the expression of genes coding phenylalanine ammonia-lyase, 4-coumarate: CoA ligase, flavanone 3-hydroxylase and flavonol synthase family members and corresponding metabolite accumulation in the phenolic biosynthesis pathway, which may positively or negatively be regulated by MYB, bHLH, WRKY, AP2/EREBP, C2C2, NAC, and C2H2 transcription factors (TFs). Furthermore, some candidate genes required for auxin, ethylene and abscisic acid signal transductions were also identified which are probably involved in berry development and flavonoid biosynthesis in response to enhanced sunlight irradiation. Taken together, this study provides a valuable overview of the light-induced phenolic metabolism and transcriptome changes, especially the dynamic responses of TFs and signaling components of phytohormones, and contributes to the further understanding of sunlight-responsive phenolic biosynthesis regulation in grape berries.

Comparison of transcriptional expression patterns of carotenoid metabolism in 'Cabernet Sauvignon' grapes from two regions with distinct climate.

The downstream flux of carotenoid metabolism in grape berries includes the biosynthesis of norisoprenoids, a group of important aroma compounds, and the production of ABA, a well-known plant hormone. This study focused on the transcriptional profiling comparison of genes participating in the biosynthesis of carotenoids, norisoprenoids, and ABA in Vitis vinifera 'Cabernet Sauvignon' grapes at pea size, veraison, and ripening stages. The grapes were obtained from Changli (CL, eastern China) and Gaotai (GT, western China) regions and analyzed using RNA-sequencing technology. The transcripts required for the carotenoid biosynthesis pathway showed a coordinated expression pattern, mainly expressing at green stage for CL and at veraison for GT, respectively. However, the carotenoid content evolution was not coincident with the timing and pattern of related gene expressions, since more carotenoids were accumulated at veraison in CL relative to two weeks before veraison in GT. Interestingly, norisoprenoid content was higher in GT than in CL, particularly at veraison and ripening, while the key gene encoding carotenoid cleavage dioxygenases, VvCCD1, showed an inverse relationship within the two regions. Higher flux was expected through the carotenoid pathway into ABA production in GT, based on the higher expression level of 9-cis-epoxycarotenoid dioxygenase and drought growing conditions. Most components involved in ABA and ethylene signaling showed distinct expression profiles in the two regions. These results revealed that downstream flux of carotenoid metabolism in grape berries showed regional differences. This study lays a foundation for future research to explore the molecular basis of climatic influences on carotenoid, norisoprenoid, and ABA biosynthesis.

Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time.

Comparison of distinct transcriptional expression patterns of flavonoid biosynthesis in Cabernet Sauvignon grapes from east and west China.

Flavonoids make a very important contribution to the organoleptic qualities of grapes and wines. In this work these were analyzed in Cabernet Sauvignon grown in Changli, Hebei Province in east China and Gaotai, Gansu Province in west China. These regions have distinctly different climates contributing to their different 'terroir'. RNA sequencing was performed to trace transcriptome changes in Cabernet Sauvignon berries at pea size, veraison and ripening, corresponding to E-L 31, 35 and 38. The accumulation of flavonols, flavan-3-ols and anthocyanins together with the expression of relevant genes were analyzed and compared between the two regions. The biosynthesis patterns were similar between two regions, but more flavonols, anthocyanins, and tri-hydroxylated flavonoids accumulated in grapes from Gaotai before berry harvest, possibly due to the higher transcript levels of the genes that encode biosynthetic enzymes and their potential candidate transcription factors. The lower levels of flavan-3-ols, mainly (-)-epigallocatechin, in the pre-veraison grapes from Changli, might be due to limited flow of carbon to the F3'5'H branch pathway, as the ratio of F3'5'H to F3'H was lower in these berries from Changli. It is suggested that the combination of climatic factors profoundly affect the flavonoid pathway in grapes from China, providing regionally specific metabolism patterns.

Effects of the sequence characteristics of miRNAs on multi-viral resistance mediated by single amiRNAs in transgenic tobacco.

Artificial microRNA (amiRNA) has become the preferred viral defence that can be induced in plants. In this study, nine amiRNA target sites were selected that were based on the sequence characteristics of natural miRNAs in the cylindrical inclusion protein (CI), nuclear inclusion a protein (NIa), nuclear inclusion b protein (NIb), and coat protein (CP) genes of Potato virus Y (PVY(N)). These amiRNAs that exhibited high similarities to the sequences of PVY(N) and TEV-SD1 were considered. To study the effectiveness of gene silencing in amiRNA-mediated viral resistance, we constructed nine amiRNA plant expression vectors by replacing the functional sequences of miRNA319a precursors with our selected amiRNA sequences. These constructs were subsequently introduced to tobacco plants. A Northern blot assay verified that the nine amiRNA plant expression vectors could successfully express amiRNAs in plants. The analysis of viral resistance demonstrated that these transgenic tobacco plants could effectively inhibit PVY(N) and TEV-SD1 viral infections. The amiRNA that targeted the NIb and CP genes displayed a higher silencing efficiency than did the amiRNAs targeted CI and NIa genes. Northern blot analysis demonstrated that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway. That is, the amiRNA and the secondary siRNA mediated the degradation of viral RNA together. Genetic analysis demonstrated that the trait for viral resistance in transgenic plants can be consistently inherited via a single copy of the transgenic sequence. Considering the correlation between the sequence characteristics and the activity of amiRNA, we concluded that a few mismatched bases between the amiRNA and the target sequence could be allowed, particularly the mismatched bases in the 3' end of the amiRNA.