Auxin alleviates cadmium toxicity by increasing vacuolar compartmentalization and decreasing long-distance translocation of cadmium in Poa pratensis.

Kentucky bluegrass (Poa pratensis L.) hyperaccumulates cadmium (Cd) and exhibits a hypertolerance. Thus, it has potential for the phytoremediation of Cd-containing soil. Auxin signaling is involved in the response to Cd stress. However, the mechanisms of auxin-mediated detoxification and Cd translocation in plants remain unclear. This study aimed to investigate the effects of exogenous application of indole-3-acetic acid (IAA) on the Cd translocation, subcellular Cd distribution, chemical forms of Cd, and transcriptional regulation of Kentucky bluegrass. The results showed that the exogenous application of IAA increased the amount of organelle-bound Cd and vacuole-compartmentalized Cd in root cells, reduced the Cd concentration in the leaf tissues (epidermis, mesophyll, and vascular bundle) and root tissues (rhizodermis and cortex) but increased in the stele, and alleviate Cd-induced leaf chlorosis and growth inhibition. The expression of genes associated with Cd transporters (ABCs, ZIPs, NASs, OPTs, and YSLs), phosphatases, oxalate decarboxylases and lignin biosynthesis were significantly regulated by exogenous IAA under Cd stress. A positive regulation of phosphatases and oxalate decarboxylases genes related to an increase in phosphate- and oxalate-bound Cd, as well as a decrease in pectate- and protein-bound Cd and inorganic Cd, thereby contributing to a decrease in Cd phytotoxicity. The significant regulation of Cd transporters associated with decreasing the long-distance translocation of Cd, and the activation of lignin biosynthesis may contribute to the development of root endodermal barriers and increase the deposition of undissolved Cd phosphates and oxalate-bound Cd in the stele. These results revealed the important role of auxin in Cd detoxification and translocation in Kentucky bluegrass and they provide a theoretical basis for the phytoremediation of Cd-containing soil.

24-epibrassinolide improves cadmium tolerance and lateral root growth associated with regulating endogenous auxin and ethylene in Kentucky bluegrass.

The application of phytohormones is a viable technique to increase the efficiency of phytoremediation in heavy metal-contaminated soils. The objective of this study was to determine how the application of 24-epibrassinolide (EBR), a brassinosteroid analog, could regulate root growth and tolerance to cadmium (Cd) stress in Kentucky bluegrass. As a result, the number of lateral root primordia and total root length in the Cd-treated seedlings decreased by 33.1 % and 56.5 %, respectively. After the application of EBR, Cd accumulation in roots and leaves, and the negative effect of Cd on root growth were reduced under Cd stress. Additionally, the expression of the brassinosteroid signaling gene PpBRI1 was significantly upregulated by exogenous EBR. Moreover, exogenous EBR upregulated the expression of genes encoding antioxidant enzymes and improved the activity of antioxidant enzymes, thereby reduced oxidative stress in roots. Finally, targeted hormonomics analysis highlighted the utility of the application of EBR to alleviate the effect of Cd on the reduction in auxin (IAA) content and the increase in ethylene (ACC) content. These were known to be associated with the upregulation in the expression of auxin biosynthesis gene PpYUCCA1 and downregulation in the expression of ethylene biosynthesis gene PpACO1 in the roots treated with Cd stress. Overall, the application of EBR alleviated Cd-induced oxidative stress in addition to improving root elongation and lateral root growth crosstalk with auxin and ethylene in Kentucky bluegrass subjected to Cd stress. This study further highlights the potential role of brassinosteroids in improving the efficiency of phytoremediation for Cd-contaminated soils.

Key factors for differential drought tolerance in two contrasting wild materials of Artemisia wellbyi identified using comparative transcriptomics.

BACKGROUND: Drought is a significant condition that restricts vegetation growth on the Tibetan Plateau. Artemisia wellbyi is a unique semi-shrub-like herb in the family Compositae, which distributed in northern and northwest of Tibetan Plateau. It is a dominant species in the community that can well adapt to virous environment stress, such as drought and low temperature. Therefore, A. wellbyi. has a potential ecological value for soil and water conservation of drought areas. Understanding the molecular mechanisms of A. wellbyi. that defense drought stress can acquire the key genes for drought resistance breeding of A. wellbyi. and provide a theoretical basis for vegetation restoration of desertification area. However, they remain unclear. Thus, our study compared the transcriptomic characteristics of drought-tolerant "11" and drought-sensitive "6" material of A. wellbyi under drought stress. RESULTS: A total of 4875 upregulated and 4381 downregulated differentially expressed genes (DEGs) were induced by drought in the tolerant material; however, only 1931 upregulated and 4174 downregulated DEGs were induced by drought in the sensitive material. The photosynthesis and transcriptional regulation differed significantly with respect to the DEGs number and expression level. We found that CDPKs (calmodulin-like domain protein kinases), SOS3 (salt overly sensitive3), MAPKs (mitogen-activated protein kinase cascades), RLKs (receptor like kinase), and LRR-RLKs (repeat leucine-rich receptor kinase) were firstly involved in response to drought stress in drought tolerant A. wellbyi. Positive regulation of genes associated with the metabolism of ABA (abscisic acid), ET (ethylene), and IAA (indole acetic acid) could play a crucial role in the interaction with other transcriptional regulatory factors, such as MYBs (v-myb avian myeloblastosis viral oncogene homolog), AP2/EREBPs (APETALA2/ethylene-responsive element binding protein family), WRKYs, and bHLHs (basic helix-loop-helix family members) and receptor kinases, and regulate downstream genes for defense against drought stress. In addition, HSP70 (heat shock protein70) and MYB73 were considered as the hub genes because of their strong association with other DEGs. CONCLUSIONS: Positive transcriptional regulation and negative regulation of photosynthesis could be associated with better growth performance under drought stress in the drought-tolerant material. In addition, the degradation of sucrose and starch in the tolerant A. wellbyi to alleviate osmotic stress and balance excess ROS. These results highlight the candidate genes that are involved in enhancing the performance of drought-tolerant A. wellbyi and provide a theoretical basis for improving the performance of drought-resistant A. wellbyi.

Triticale Improvement: Mining of Genes Related to Yellow Rust Resistance in Triticale Based on Transcriptome Sequencing.

Yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (Pst) is a major destructive fungal disease of small grain cereals, leading to large yield losses. The breeding of resistant varieties is an effective, sustainable way to control yellow rust. Elucidation of resistance mechanisms against yellow rust and identification of candidate genes associated with rust resistance are thus crucial. In this study, seedlings of two Triticosecale Wittmack cultivars, highly resistant Gannong No. 2 and susceptible Shida No. 1, were inoculated with Pst race CYR34. Transcriptome sequencing (RNA-seq) was then used to investigate their transcriptional responses against pathogen infection before and after the appearance of symptoms-10 and 20 days after inoculation, respectively. According to the RNA-seq data, the number of upregulated and downregulated differentially expressed genes (DEGs) in the resistant cultivar was greater than in the susceptible cultivar. A total of 2,560 DEGs commonly expressed in the two cultivars on two sampling dates were subjected to pathway analysis, which revealed that most DEGs were closely associated with defense and metabolic activities. Transcription factor enrichment analysis indicated that the expressions of NAC, WRKY, and FAR1 families were also significantly changed. Further in-depth analysis of resistance genes revealed that almost all serine/threonine-protein kinases were upregulated in the resistant cultivar. Other genes related to disease resistance, such as those encoding disease-resistance- and pathogenesis-related proteins were differentially regulated in the two cultivars. Our findings can serve as a resource for gene discovery and facilitate elucidation of the complex defense mechanisms involved in triticale resistance to Pst.

Transcriptional Regulation of Different Rhizome Parts Reveal the Candidate Genes That Regulate Rhizome Development in Poa pratensis.

A strong rhizome can enhance the ability of a plant to resist drought, low temperature, and other stresses, as it can help plants rapidly obtain water and nutrients. Poa pratensis var. anceps Gaud. cv. Qinghai (QH) is a variant of P. pratensis that is widely distributed in natural grasslands above 3000 m above sea level on the Qinghai-Tibet Plateau. It forms turf easily and has strong soil-fixing ability due to its well-developed rhizomes. Understanding the molecular mechanism of rhizome development in this species is essential for cultivating new varieties of rhizome-type pasture for ecological protection. To clarify the transcriptional regulatory changes in different parts of the rhizome, we analyzed three different rhizome parts (rhizome buds, rhizome nodes, and rhizome internodes) of QH and weak-rhizome wild P. pratensis material (SN) using RNA sequencing. A total of 3806 genes were specifically expressed in Q_B, 1104 genes were specifically expressed in Q_N, and 1181 genes were specifically expressed in Q_I. Analysis showed that MYB, B3, NAC, BBR-BPC, AP2 MIKC_MADS, BSE1, and C2H2 may be key transcription factors regulating rhizome development. These genes interacted with multiple functional genes related to carbohydrate, secondary metabolism, and signal transduction, thus ensuring the normal development of the rhizomes. In particular, SUS (sucrose synthase) [EC:2.4.1.13] is specifically expressed in Q_I, which may be an inducing factor for the production of new plants from Q_B and Q_N. Additionally, PYL, PP2C, and SNRK2, which are involved in the abscisic acid signaling pathway, were differentially expressed in Q_N. In addition, genes related to protein modification and degradation, such as CIPKs, MAPKs, E2, and E3 ubiquitin ligases, were also involved in rhizome development. This study laid a foundation for further functional genomics studies on rhizome development in P. pratensis.

Cadmium stress suppresses the tillering of perennial ryegrass and is associated with the transcriptional regulation of genes controlling axillary bud outgrowth.

Perennial ryegrass (Lolium perenne L.), a grass species with superior tillering capacity, plays a potential role in the phytoremediation of cadmium (Cd)-contaminated soils. Tiller production is inhibited in response to serious Cd stress. However, the regulatory mechanism of Cd stress-induced inhibition of tiller development is not well documented. To address this issue, we investigated the phenotype, the expression levels of genes involved in axillary bud initiation and bud outgrowth, and endogenous hormone biosynthesis and signaling pathways in seedlings of perennial ryegrass under Cd stress. The results showed that the number of tillers and axillary buds in the Cd-treated seedlings decreased by 67% and 21%, respectively. The suppression of tiller production in the Cd-treated seedlings was more closely associated with the inhibition of axillary bud outgrowth than with bud initiation. Cd stress upregulated the expression level of genes related to axillary bud dormancy and downregulated bud activity genes. Additionally, genes involved in strigolactone biosynthesis and signaling, auxin transport and signaling, and cytokinin degradation were upregulated in Cd-treated seedlings, and cytokinin biosynthesis gene expression were decreased by Cd stress. The content of zeatin in the Cd-treated pants was significantly reduced by 69~85% compared to the control plants. The content of indole-3-acetic acid (IAA) remains constant under Cd stress. Overall, Cd stress induced axillary bud dormancy and subsequently inhibited axillary bud outgrowth. The decrease of zeatin content and upregulation of genes involved in strigolactone signaling and bud dormancy might be responsible for the inhibition of axillary bud outgrowth.

Identification and Expression Analysis of the SWEET Gene Family from Poa pratensis Under Abiotic Stresses.

The sugars will eventually be exported transporters (SWEET) gene family is a glycoprotein gene family that can regulate the transport of sugar in plants and plays an important role in plant growth and development, as well as in response to environmental stress. In this study, Kentucky bluegrass (cv. Baron) seedlings were grown in various treatments, including heavy metal cadmium, salt, drought, cold, and heat stress for 6 h, 24 h, 48 h, and 7 day. The relative expression of the identified PpSWEET genes in Kentucky bluegrass was measured. The results showed there were a total of 13 SWEET genes, which could be divided into four clades by phylogenetic analysis. Most PpSWEET genes are alkali proteins with seven transmembrane helices. Moreover, almost all PpSWEET proteins possess similar conserved motifs and active sites. In addition, an analysis of the relative expression of PpSWEET genes under various stress treatments indicated that PpSWEET12 and PpSWEET15 had very high expression under the five types of stress, meaning they can be used as important candidate genes for studying responses to environmental stresses of turfgrass. Furthermore, certain genes only showed changes in expression under one or two specific stress treatments. This study provides important insight into the SWEET gene family in Kentucky bluegrass and its functional roles in responses to various environmental stresses.

Transcriptional regulation and expression network responding to cadmium stress in a Cd-tolerant perennial grass Poa Pratensis.

Kentucky bluegrass has good capability to absorb and accumulate cadmium (Cd) through developed root system, thus having potential phytoremediation function in Cd contaminated soils. Understanding the molecular mechanisms of Cd tolerance and accumulation in this species will be crucial to generating novel Cd-tolerance cultivars through genetic improvement, while it has not well documented yet. In the present study, comparative transcriptome analysis was performed for the seedlings of high Cd-tolerant genotype (M) and low Cd-tolerant genotype (R) under Cd stress. A total of 7022 up-regulated and 1033 down-regulated transcripts were identified in M genotype, whereas, only 850 up-regulated and 846 down-regulated transcripts were detected in R. Further transcriptional regulation analysis in M genotype showed that Dof, MADS25, BBR-BPC, B3, bZIP23 and MYB30 might be the hub transcription factors in response to Cd stress due to the orchestrated multiple functional genes associated with carbohydrate, lipid and secondary metabolism, as well as signal transduction. Differential expressed genes involved in auxin, ethylene, brassinosteroid and ABA signalling formed signal transduction cascades, which interacted with hub transcription factors, thereby finally orchestrated the expression of multiple genes associated with cell wall and membrane stability, cell elongation and Cd tolerance, including IAAs, ARFs, SnRK2, PP2C, PIFs, BES1/BZR1, CCR, CAD, FATB, fabF and HACD. Additionally, post-transcriptional modification of CIPKs, MAPKs, WAXs, UBCs, and E3 ubiquitin ligases were identified and also involved in plant signalling pathways and abiotic resistance. The study could contribute to our understanding the transcriptional regulation and complex internal network associated with Cd tolerance in Kentucky bluegrass.

Transcriptional Responses of Creeping Bentgrass to 2,3-Butanediol, a Bacterial Volatile Compound (BVC) Analogue.

Bacterial volatile compounds (BVCs) have been reported to enhance plant growth and elicit plant defenses against fungal infection and insect damage. The objective of this study was to determine transcriptomic changes in response to synthetic BVC that could be associated with plant resistance to Rhizoctonia solani in creeping bentgrass. The 2,3-butanediol (BD) (250 µM) was sprayed on creeping bentgrass leaves grown in jam jars. The result showed that synthetic BD induced plant defense against R. solani for creeping bentgrass. Transcriptomic analysis demonstrated that more genes were repressed by BD while less showed up-regulation. BD suppressed the expression of some regular stress-related genes in creeping bentgrass, such as pheromone activity, calcium channel activity, photosystem II oxygen evolving complex, and hydrolase activity, while up-regulated defense related transcription factors (TFs), such as basic helix-loop-helix (bHLH) TFs, cysteine2-cysteine2-contans-like (C2C2-CO) and no apical meristem TFs (NAC). Other genes related to disease resistance, such as jasmonic acid (JA) signaling, leucine rich repeats (LRR)-transmembrane protein kinase, pathogen-related (PR) gene 5 receptor kinase and nucleotide binding site-leucine rich repeats (NBS-LRR) domain containing plant resistance gene (R-gene) were also significantly up-regulated. These results suggest that BD may induce changes to the plant transcriptome in induced systemic resistance (ISR) pathways.

5-Aminolevulinic acid modulates antioxidant defense systems and mitigates drought-induced damage in Kentucky bluegrass seedlings.

Drought stress occurs frequently and severely as a result of global climate change, and it exerts serious effects on plants. 5-Aminolevulinic acid (5-ALA) plays a crucial role in conferring abiotic stress tolerance in plants. To enhance the drought tolerance of turfgrass and investigate the effects of 5-ALA on antioxidant metabolism and gene expression under drought stress conditions, exogenous 5-ALA was applied by foliar spraying before Kentucky bluegrass (Poa pratensis L.) seedlings were exposed to drought [induced by 10% polyethylene glycol (PEG)] stress for 20 days. 5-ALA pretreatment increased turf quality (TQ) and leaf relative water content (RWC) while reducing reactive oxygen species (ROS) production including H2O2 content and O2•- generation rate, lipoxygenase (LOX) activity, and malondialdehyde (MDA) content under drought stress. 5-ALA pretreatment maintained ascorbate (AsA) and glutathione (GSH) contents and the ASA/DHA and GSH/GSSG ratios at high levels, and it enhanced the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), which are crucial for scavenging drought-induced ROS. In addition, 5-ALA upregulated the relative expression levels of Cu/ZnSOD, APX, GPX, and DHAR but downregulated those of CAT and GR under drought stress. These results indicated that the application of 5-ALA might improve turfgrass quality and promote drought tolerance in Kentucky bluegrass through reducing oxidative damage and increasing non-enzyme antioxidant levels and antioxidant enzyme activity at transcriptional and posttranscriptional levels.

Selection of Candidate Reference Genes for Gene Expression Analysis in Kentucky Bluegrass (Poa pratensis L.) under Abiotic Stress.

Kentucky bluegrass (Poa pratensis L.) belong to Gramineae and is widely used in lawns, golf courses, landscapes, and sport fields as a prominent cool-season grass. Gene expression patterns during different stages of plant development can provide clues toward the understanding of its biological functions. The selection and validation of reference genes are the first steps in any real-time quantitative PCR gene expression study. Therefore, suitable reference genes are necessary for obtaining reliable results in real-time quantitative PCR analyses of Kentucky bluegrass. In the present study, 9 candidate reference genes were chosen, and their expression stability in the leaves and roots of Kentucky bluegrass under different stresses (drought, salt, heat, and cold) were evaluated using the GeNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the experimental conditions. The combination of SAM with GAPDH was the most stable in leaves under salt stress and cold stress, while TUB combined with ACT or GAPDH was stable in roots under salt or cold stress, respectively. ACT and SAM maintained stable expression in drought-treated leaves, and GAPDH combined with ACT was stable in drought-treated roots. SAM and TUB exhibited stable expression in heat-treated leaves. ACT and RPL were stable in heat-treated roots. In addition, the expression patterns of PpFEH in response to drought and cold stress were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause erroneous results. This work is the first study on the expression stability of reference genes in Kentucky bluegrass and will be particularly useful in the selection of stress-tolerance genes and the identification of the molecular mechanisms conferring stress tolerance in this species.