A novel TF molecular switch-mechanism found in two contrasting ecotypes of a psammophyte, Agriophyllum squarrosum, in regulating transcriptional drought memory.

BACKGROUND: Prior drought stress may change plants response patterns and subsequently increase their tolerance to the same condition, which can be referred to as "drought memory" and proved essential for plants well-being. However, the mechanism of transcriptional drought memory in psammophytes remains unclear. Agriophyllum squarrosum, a pioneer species on mobile dunes, is widely spread in Northern China's vast desert areas with outstanding ability of water use efficiency. Here we conducted dehydration-rehydration treatment on A. squarrosum semi-arid land ecotype AEX and arid land ecotype WW to dissect the drought memory mechanism of A. squarrosum, and to determine the discrepancy in drought memory of two contrasting ecotypes that had long adapted to water heterogeneity. RESULT: Physiological traits monitoring unveiled the stronger ability and longer duration in drought memory of WW than that of AEX. A total of 1,642 and 1,339 drought memory genes (DMGs) were identified in ecotype AEX and WW, respectively. Furthermore, shared DMGs among A. squarrosum and the previously studied species depicted that drought memory commonalities in higher plants embraced pathways like primary and secondary metabolisms; while drought memory characteristics in A. squarrosum were mainly related to response to heat, high light intensity, hydrogen peroxide, and dehydration, which might be due to local adaptation to desert circumstances. Heat shock proteins (HSPs) occupied the center of the protein-protein interaction (PPI) network in drought memory transcription factors (TF), thus playing a key regulatory role in A. squarrosum drought memory. Co-expression analysis of drought memory TFs and DMGs uncovered a novel regulating module, whereby pairs of TFs might function as molecular switches in regulating DMG transforming between high and low expression levels, thus promoting drought memory reset. CONCLUSION: Based on the co-expression analysis, protein-protein interaction prediction, and drought memory metabolic network construction, a novel regulatory module of transcriptional drought memory in A. squarrosum was hypothesized here, whereby recurrent drought signal is activated by primary TF switches, then amplified by secondary amplifiers, and thus regulates downstream complicated metabolic networks. The present research provided valuable molecular resources on plants' stress-resistance basis and shed light on drought memory in A. squarrosum.

Corrigendum: Integrated metabolomics and transcriptomics insights on flavonoid biosynthesis of a medicinal functional forage, Agriophyllum squarrosum (L.), based on a common garden trial covering six ecotypes.

[This corrects the article DOI: 10.3389/fpls.2022.985572.].

Integrated metabolomics and transcriptomics insights on flavonoid biosynthesis of a medicinal functional forage, Agriophyllum squarrosum (L.), based on a common garden trial covering six ecotypes.

Agriophyllum squarrosum (L.) Moq., well known as sandrice, is an important wild forage in sandy areas and a promising edible and medicinal resource plant with great domestication potential. Previous studies showed flavonoids are one of the most abundant medicinal ingredients in sandrice, whereby isorhamnetin and isorhamnetin-3-glycoside were the top two flavonols with multiple health benefits. However, the molecular regulatory mechanisms of flavonoids in sandrice remain largely unclear. Based on a common garden trial, in this study, an integrated transcriptomic and flavonoids-targeted metabolomic analysis was performed on the vegetative and reproductive periods of six sandrice ecotypes, whose original habitats covered a variety of environmental factor gradients. Multiple linear stepwise regression analysis unveiled that flavonoid accumulation in sandrice was positively correlated with temperature and UVB and negatively affected by precipitation and sunshine duration, respectively. Weighted co-expression network analysis (WGCNA) indicated the bHLH and MYB transcription factor (TF) families might play key roles in sandrice flavonoid biosynthesis regulation. A total of 22,778 differentially expressed genes (DEGs) were identified between ecotype DL and ecotype AEX, the two extremes in most environmental factors, whereby 85 DEGs could be related to known flavonoid biosynthesis pathway. A sandrice flavonoid biosynthesis network embracing the detected 23 flavonoids in this research was constructed. Gene families Plant flavonoid O-methyltransferase (AsPFOMT) and UDP-glucuronosyltransferase (AsUGT78D2) were identified and characterized on the transcriptional level and believed to be synthases of isorhamnetin and isorhamnetin-3-glycoside in sandrice, respectively. A trade-off between biosynthesis of rutin and isorhamnetin was found in the DL ecotype, which might be due to the metabolic flux redirection when facing environmental changes. This research provides valuable information for understanding flavonoid biosynthesis in sandrice at the molecular level and laid the foundation for precise development and utilization of this functional resource forage.

Transcriptome profiling reveals that foliar water uptake occurs with C3 and crassulacean acid metabolism facultative photosynthesis in Tamarix ramosissima under extreme drought.

Tamarix ramosissima is a typical desert plant species that is widely distributed in the desert areas of Northwest China. It plays a significant role in sand fixation and soil water conservation. In particular, how it uses water to survive in the desert plays an important role in plant growth and ecosystem function. Previous studies have revealed that T. ramosissima can alleviate drought by absorbing water from its leaves under extreme drought conditions. To date, there is no clear molecular regulation mechanism to explain foliar water uptake (FWU). In the present study, we correlated diurnal meteorological data, sap flow and photosynthetic parameters to determine the physical and biological characteristics of FWU. Our results suggested that the lesser the groundwater, the easier it is for T. ramosissima to absorb water via the leaves. Gene ontology annotation and Kyoto Encyclopaedia of Genes and Genomes pathway analysis of the transcriptome profile of plants subjected to high humidity suggested that FWU was highly correlated to carbohydrate metabolism, energy transfer, pyruvate metabolism, hormone signal transduction and plant-pathogen interaction. Interestingly, as a C3 plant, genes such as PEPC, PPDK, MDH and RuBP, which are involved in crassulacean acid metabolism (CAM) photosynthesis, were highly upregulated and accompanied by FWU. Therefore, we proposed that in the case of sufficient water supply, C3 photosynthesis is used in T. ramosissima, whereas in cases of extreme drought, starch is degraded to provide CO2 for CAM photosynthesis to make full use of the water obtained via FWU and the water that was transported or stored to assimilating branches and stems. This study may provide not only an important theoretical foundation for FWU and conversion from C3 plants to CAM plants but also for engineering improved photosynthesis in high-yield drought-tolerant plants and mitigation of climate change-driven drought.

Variations in Flavonoid Metabolites Along Altitudinal Gradient in a Desert Medicinal Plant Agriophyllum squarrosum.

Agriophyllum squarrosum (L.) Moq., a pioneer plant endemic to the temperate deserts of Asia, could be domesticated into an ideal crop with outstanding ecological and medicinal characteristics. A previous study showed differential flavonoid accumulation between two in situ altitudinal ecotypes. To verify whether this accumulation was determined by environmental or genetic factors, we conducted flavonoid-targeted metabolic profiling among 14 populations of A. squarrosum collected from regions with different altitudes based on a common garden experiment. Results showed that the most abundant flavonoid in A. squarrosum was isorhamnetin (48.40%, 557.45 µg/g), followed by quercetin (13.04%, 150.15 µg/g), tricin (11.17%, 128.70 µg/g), isoquercitrin (7.59%, 87.42 µg/g), isovitexin (7.20%, 82.94 µg/g), and rutin (7.00%, 80.62 µg/g). However, based on a common garden at middle-altitude environment, almost none of the flavonoids was enriched in the high-altitude populations, and even some flavonoids, such as quercetin, tricin, and rutin, were significantly enriched in low-altitude populations. This phenomenon indicated that the accumulation of flavonoids was not a result of local adaptation to high altitude. Furthermore, association analysis with in situ environmental variables showed that the contents of quercetin, tricin, and rutin were strongly positively correlated with latitude, longitude, and precipitation gradients and negatively correlated with temperature gradients. Thus, we could conclude that the accumulations of flavonoids in A. squarrosum were more likely as a result of local adaption to environmental heterogeneity combined with precipitation and temperature other than high altitude. This study not only provides an example to understand the molecular ecological basis of pharmacognosy, but also supplies methodologies for developing a new industrial crop with ecological and agricultural importance.

Organic acid metabolites involved in local adaptation to altitudinal gradient in Agriophyllum squarrosum, a desert medicinal plant.

Agriophyllum squarrosum (L.) Moq., a pioneer plant endemic to the temperate deserts of Asia, could be domesticated into an ideal crop with outstanding ecological and medicinal characteristics. A previous study showed differential organic acid accumulation between two in situ altitudinal ecotypes. To verify whether this accumulation was determined by environmental or genetic factors, we conducted organic acid targeted metabolic profiling among 14 populations of A. squarrosum collected from regions with different altitudes based on a common garden experiment. Results showed that the most abundant organic acid in A. squarrosum was citric acid (96.03%, 2322.90 µg g-1). Association analysis with in situ environmental variables showed that salicylic acid content was positively correlated with altitudinal gradient. Considering the enrichment of salicylic acid and protocatechualdehyde in high-altitude populations based on the common garden experiment, and the high expression of their biosynthesis relative genes (i.e., PAL and C4H) in the in situ high-altitude ecotype, we propose that organic acid accumulation could be involved in local adaptation to high altitudes. This study not only addresses the molecular basis of local adaptation involving the accumulation of organic acids in the desert plant A. squarrosum but also provides a method to screen wild germplasms to mitigate the impact of global climate change.

Genomic Adaptive Evolution of Sand Rice (Agriophyllum squarrosum) and Its Implications for Desert Ecosystem Restoration.

Natural selection is a significant driver of population divergence and speciation of plants. Due to local adaptation to geographic regions with ecological gradients, plant populations harbored a wide range of adaptive genetic variation to enable them to survive the heterogeneous habitats. This is all the more necessary for desert plants, as they must tolerant more striking gradients of abiotic stresses. However, the genomic mechanism by which desert plants adapt to ecological heterogeneity remains unclear, which could help to guide the sustainability of desert ecosystems. Here, using restriction-site-associated DNA sequencing in 38 natural populations, we investigated the genomic divergence and environmental adaptation of sand rice, Agriophyllum squarrosum, an annual pioneer species that covers sand dunes in northern China. Population genetic structure analyses showed that sand rice could be divided into three geographically distinct lineages, namely, Northwest, Central, and East. Phylogeographic analyses revealed that the plant might originate locally in Bergen County and further differentiated into the East lineage and then the Central lineage. Ecological niche modeling found that different lineages occupied distinct ecological niches, suggesting that the ecological gradient would have triggered genomic differentiation among sand rice lineages. Ecological association study supported that the three SNPs under divergent selection were closely correlated with precipitation gradients, indicating that precipitation might be the most important stress trigger for lineage diversity in sand rice. These adaptive SNPs could be used to genotype suitable germplasms for the ecological restoration of specific desertified lands. Further analyses found that genetic structure could significantly overestimate the signals for balancing selection. Within the Central lineage, we still found that 175 SNPs could be subject to balancing selection, which could be the means by which sand rice maintains genetic diversity and adapts to multiple stresses across heterogeneous deserts and sandy lands. From a genomic point of view, this study highlighted the local and global adaptation patterns of a desert plant to extreme and heterogeneous habitats. Our data provide molecular guidance for the restoration of desertified lands in the arid and semi-arid regions of China and could facilitate the marker assistant breeding of this potential crop to mitigate climate change.

Leaf size variations in a dominant desert shrub, Reaumuria soongarica, adapted to heterogeneous environments.

The climate in arid Central Asia (ACA) has changed rapidly in recent decades, but the ecological consequences of this are far from clear. To predict the impacts of climate change on ecosystem functioning, greater attention should be given to the relationships between leaf functional traits and environmental heterogeneity. As a dominant constructive shrub widely distributed in ACA, Reaumuria soongarica provided us with an ideal model to understand how leaf functional traits of desert ecosystems responded to the heterogeneous environments of ACA. Here, to determine the influences of genetic and ecological factors, we characterized species-wide variations in leaf traits among 30 wild populations of R. soongarica and 16 populations grown in a common garden. We found that the leaf length, width, and leaf length to width ratio (L/W) of the northern lineage were significantly larger than those of other genetic lineages, and principal component analysis based on the in situ environmental factors distinguished the northern lineage from the other lineages studied. With increasing latitude, leaf length, width, and L/W in the wild populations increased significantly. Leaf length and L/W were negatively correlated with altitude, and first increased and then decreased with increasing mean annual temperature (MAT) and mean annual precipitation (MAP). Stepwise regression analyses further indicated that leaf length variation was mainly affected by latitude. However, leaf width was uncorrelated with altitude, MAT, or MAP. The common garden trial showed that leaf width variation among the eastern populations was caused by both local adaptation and phenotypic plasticity. Our findings suggest that R. soongarica preferentially changes leaf length to adjust leaf size to cope with environmental change. We also reveal phenotypic evidence for ecological speciation of R. soongarica. These results will help us better understand and predict the consequences of climate change for desert ecosystem functioning.

Transcriptomes Divergence of Ricotia lunaria Between the Two Micro-Climatic Divergent Slopes at "Evolution Canyon" I, Israel.

As one of the hotspot regions for sympatric speciation studies, Evolution Canyon (EC) became an ideal place for its high level of microclimatic divergence interslopes. In this study, to highlight the genetic mechanisms of sympatric speciation, phenotypic variation on flowering time and transcriptomic divergence were investigated between two ecotypes of Ricotia lunaria, which inhabit the opposite temperate and tropical slopes of EC I (Lower Nahal Oren, Mount Carmel, Israel) separated by 100 m at the bottom of the slopes. Growth chamber results showed that flowering time of the ecotype from south-facing slope population # 3 (SFS 3) was significantly 3 months ahead of the north-facing slope population # 5 (NFS 5). At the same floral development stage, transcriptome analysis showed that 1,064 unigenes were differentially expressed between the two ecotypes, which enriched in the four main pathways involved in abiotic and/or biotic stresses responses, including flavonoid biosynthesis, α-linolenic acid metabolism, plant-pathogen interaction and linoleic acid metabolism. Furthermore, based on Ka/Ks analysis, nine genes were suggested to be involved in the ecological divergence between the two ecotypes, whose homologs functioned in RNA editing, ABA signaling, photoprotective response, chloroplasts protein-conducting channel, and carbohydrate metabolism in Arabidopsis thaliana. Among them, four genes, namely, SPDS1, FCLY, Tic21 and BGLU25, also showed adaptive divergence between R. lunaria and A. thaliana, suggesting that these genes could play an important role in plant speciation, at least in Brassicaceae. Based on results of both the phenotype of flowering time and comparative transcriptome, we hypothesize that, after long-time local adaptations to their interslope microclimatic environments, the molecular functions of these nine genes could have been diverged between the two ecotypes. They might differentially regulate the expression of the downstream genes and pathways that are involved in the interslope abiotic stresses, which could further diverge the flowering time between the two ecotypes, and finally induce the reproductive isolation establishment by natural selection overruling interslope gene flow, promoting sympatric speciation.

A whole-transcriptome approach to evaluate reference genes for quantitative diurnal gene expression studies under natural field conditions in Tamarix ramosissima leaves

Background: Tamarix ramosissima is a desert forest tree species that is widely distributed in the drought-stricken areas to sustain the fragile ecosystem. Owing to its wide usage in the desert restoration of Asia, it can be used as an ecophysiological model plant. To obtain reliable and accurate results, a set of reference genes should be screened before gene expression. However, up to date, systematical evaluation of reference genes has not been conducted in T. ramosissima. Results: In this study, we used eigenvalues derived from principal component analysis to identify stable expressed genes from 72,035 unigenes from diurnal transcriptomes under natural field conditions. With combined criteria of read counts above 900 and CV of FPKM below 0.3, a total of 7385 unigenes could be qualified as candidate reference genes in T. ramosissima. By using three statistical algorithm packages, geNorm, NormFinder, and BestKeeper, the stabilities of these novel reference genes were further compared with a panel of traditional reference genes. The expression patterns of three aquaporins (AQPs) suggested that at least UBQ (high expression), EIF4A2 (low expression), and GAPDH (moderate expression) could be qualified as ideal reference genes in both RT-PCR and RNA-seq analysis of T. ramosissima. Conclusions: This work will not only facilitate future studies on gene expression and functional analysis of genetic resources of desert plants but also improve our understanding of the molecular regulation of water transport in this plant, which could provide a new clue to further investigate the drought adaptation mechanism of desert plant species under harsh environments.

Development of highly polymorphic simple sequence repeat markers using genome-wide microsatellite variant analysis in Foxtail millet [Setaria italica (L.) P. Beauv].

BACKGROUND: Foxtail millet (Setaria italica (L.) Beauv.) is an important gramineous grain-food and forage crop. It is grown worldwide for human and livestock consumption. Its small genome and diploid nature have led to foxtail millet fast becoming a novel model for investigating plant architecture, drought tolerance and C4 photosynthesis of grain and bioenergy crops. Therefore, cost-effective, reliable and highly polymorphic molecular markers covering the entire genome are required for diversity, mapping and functional genomics studies in this model species. RESULT: A total of 5,020 highly repetitive microsatellite motifs were isolated from the released genome of the genotype 'Yugu1' by sequence scanning. Based on sequence comparison between S. italica and S. viridis, a set of 788 SSR primer pairs were designed. Of these primers, 733 produced reproducible amplicons and were polymorphic among 28 Setaria genotypes selected from diverse geographical locations. The number of alleles detected by these SSR markers ranged from 2 to 16, with an average polymorphism information content of 0.67. The result obtained by neighbor-joining cluster analysis of 28 Setaria genotypes, based on Nei's genetic distance of the SSR data, showed that these SSR markers are highly polymorphic and effective. CONCLUSIONS: A large set of highly polymorphic SSR markers were successfully and efficiently developed based on genomic sequence comparison between different genotypes of the genus Setaria. The large number of new SSR markers and their placement on the physical map represent a valuable resource for studying diversity, constructing genetic maps, functional gene mapping, QTL exploration and molecular breeding in foxtail millet and its closely related species.