Environment effect analysis –cold/freezing/drought

Low temperature during early spring, late autumn, and winter is a primary quality and productivity affecting factor for chrysanthemum. Ren et al. (2014) generated six separate RNA-Seq libraries (A: cold acclimation (CA) 4°C for one week, B: freezing treatment without prior CA -5°C for 1h and C: freezing treatment without prior CA -

58 5°C for 2 h, D: freezing treatments with prior CA of 4°C for one week, followed by −5°C for 1 E: freezing treatments with prior CA of 4°C for one week, followed by −5°C for 2 h and F: the control) from leaves and stem. Overall, from library A to library F, 7,270,059, 7,052,023, 7,013,186, 7,228,380, 7,299,665, and 7,468,883 clean reads were obtained respectively. The proportion of clearly mapped reads per library ranged from 77.13% to 81.03% in the libraries. A large number of differential transcribed genes (DTGs) were identified in this study and indicated that more DTGs were identified in the treatments that underwent a prior CA stage than those which had not experienced a CA stage. Moreover, a large amount of unigenes were obtained with KEGG annotation and the major pathways were detected including “metabolism”, “biosynthesis of secondary metabolites”, “hormone signal transduction”, etc. related with cold tolerance. The important detections were confirmed by qPCR also. Finally, the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance were confirmed in chrysanthemum(Ren et al. 2014). In Ammopiptanthus mongolicus studies, the seedlings were untreated or treated with drought or, cold separately (Wu et al. 2014). For de novo transcriptome sequencing and assembly, treatments were as follows: Cold was applied as a gradual cooling process under a 16 h dim light/8 h dark cycle. The cooling process started at 4°C and moved to -8°C over a 48 hour period during which time samples were taken. The drought treatments used the same time points (up to 48 h of dehydration) as cold stress. For transcriptome profile analysis seedlings were sampled at 2, 8 and 24 h after the initiation of the cold or drought treatment respectively. 86,058 unigenes were assembled and 51,014 unigenes had an annotated function and 2,440 encoded transcription factors (TFs). Among the results, 2,028 and 2,026 DEGs were common across the three time points of drought and cold treatments respectively, and 971 DEGs were co-regulated by both stresses. 26,999 of the 86,058 unigenes were assigned to 125 KEGG pathways and “Metabolic pathways” was the most important. Moreover, ABA (abscisic acid), BR (brassinosteroid) and ethylene signaling pathways were found to be the main hormone pathways for plants stress response. In addition, the flavonoid biosynthesis genes were enriched in the DEGs co-up-regulated by both stresses and membrane protein genes and genes related to chloroplast were abundant, specifically up-regulated by drought or cold, respectively (Wu et al. 2014).

The fresh leaves, stems, bulbs, and roots of Lilium lancifolium, an important cold- resistant wild flower for lily cold resistance breeding, were mixed cold treated for 0, 2

59 h or 16 h at 4 °C (Wang et al. 2014). Using RNA-Seq analysis, about 104,703 million clean paired-end reads (90 bp) were obtained from the three libraries. Through use of BLAST analysis with the Swiss-Prot protein database, 18,736 unigenes were obtained. In addition, 25 COG categories were found which formed 12 SOM clusters. The results showed that cold signal transduction genes such as LlICE and LlCDPK and transcription factor genes such as LlDREB1/CBF, LlAP2/EREBP, LlNAC1, LlR2R3- MYB and LlBZIP were expressed highly in the bulb after cold treatment. LlFAD3, Llβ- amylase, LlP5CS and LlCLS cold related genes were found to be present with cold treatment and these take part in cellular osmoprotection and carbohydrate metabolism (Wang et al. 2014).

Chorispora bungeana, a perennial subnival alpine plant, has strong cold tolerance mechanisms. In order to obtain more information, seedlings were analyzed by Illumina

deep-sequencing and compared with Arabidopsis. From 7 day old seedlings 10 random

plants were selected (roots, shoots and leaves) and subjected to 24 h chilling stress then

sampled. After RNA-Seq analysis, a total of 54,870 unigenes were obtained by de novo

assembly, with 3,484 chilling up-regulated and 4,571 down-regulated. When C. bungeana was compared with Arabidopsis, some functional networks of biological processes were the same, such as cold response and molecular functions. The results

showed the generally cold related genes CBF2 and CBF3 were not found to be up-

regulated in C. bungeana. In addition, Karrikins were identified as new plant growth

regulators involved in chilling responses of both C. bungeana and Arabidopsis.

Moreover, up-regulated genes related to protein phosphorylation and auto-

ubiquitination processes were over-represented in both species (Zhao et al. 2012).

In Anthurium andraeanum, a popular tropical flower, over two billion bases of

high-quality sequence and 44,382 unigenes (mean length=560 bp) were produced by Illumina sequencing technology. 27,396 unigenes were functionally annotated (E-value

of 10-5). Among the 4,363 identified DGEs, 292, 805 and 708 genes were up-regulated

after1-h, 5-h and 24-h cold treatment, respectively. For 1-h cold treatment, the most enriched pathways were: the photosynthesis pathway, metabolic pathways and oxidative phosphorylation pathways. For the 5-h cold treatment, the metabolic pathways and oxidative phosphorylation pathways were significantly up-regulated. After 24-h cold treatment, the mRNA surveillance pathway, RNA transport pathway and plant-pathogen interaction pathway were significantly enriched. In addition, a total

60 of 39 cold-inducible transcription factors, including subsets of AP2/ERF, Zinc figure,

NAC, MYB and bZIP family members were identified (Tian et al. 2013b).