Alternate wetting and drying (AWD) irrigation has been widely used as an efficient rice production method to obtain better yield without continuous flooding (CF) of the paddy field. However, how this practice affects gene expression to regulate rice physiology and morphology is largely unknown. In this study, we used two rice varieties, Nipponbare, a lowland rice cultivar, and Gaoshan 1, an upland cultivar, and found that root dry weight (RDW) and root oxidation activity (ROA) in both cultivars

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... substantially increased in response to AWD. We then analyzed the differences in transcriptome profiles of their roots irrigated in AWD vs. CF conditions. AWD responsive genes are mainly involved in lignin biosynthetic pathway and phytohormone signal transduction pathway and belong mainly to bHLH, bZIP, NAC, WRKY, and HSF transcription factor families. We discussed how these differentially expressed genes may contribute to the morphological adaptations observed in roots exposed to AWD. This analysis also provides useful information to explain the similarities and differences in adaptation to AWD irrigation between the two rice ecotypes. Introduction As one of the earliest domesticated food crops, rice (Oryza sativa L.) is an important staple food for half of the world population [1, 2] . China shares about 19% of the global rice planting area and contributes to 32% of the global rice production (FAO, http://www.fao.org/faostat/zh/#data), and the amount of rice irrigation water accounts for 65% of annual agricultural water consumption in China [3] . Unfortunately, large parts of China face either physical or economic water scarcity [4] . Moreover, agricultural irrigation water will gradually and rapidly deplete due to fierce demand for water resources from urban and industrial sectors, and with increasing global commercialization it seems that industry will receive priority over irrigation [5] . In China, over 95% of rice is grown under traditional continuous flooding (CF) irrigation which expends a large amount of labor, time and * Corresponding authors: Weifeng Xu, Journal Pre-proof J o u r n a l P r e -p r o o f 2 energy due to the increased pumping of water in flooded fields [6] . Alternate wetting and drying (AWD) irrigation has been developed as a novel water-saving technique and has been adopted in many countries such as China, Bangladesh, India and Vietnam [7, 8] . By reducing the required number of irrigation events, AWD irrigation can reduce water consumption by up to 30%-35% in comparison to CF irrigation [9,10]. Among rice varieties, while lowland rice is grown in deep water irrigation and plants possess shallow-thin roots, upland rice cultivation involves dry fields and plants acquire deeper thicker roots [11, 12] . Upland rice varieties have evolved to adapt to the drought-prone regions with increased drought tolerance due to the long-term natural and human selection [13] . Thus, global water shortage has forced farmers to go on with upland rice cultivation. The underlying genetics behind this evolution of upland rice from lowland varieties is currently unexplored, except for some transcriptome and few quantitative trait loci (QTL) mapping studies [14] [15] [16] . In a cross between shallow-rooted lowland and deep-rooted upland variety, QTL were searched for root growth and were mapped to (DRO1, QRO1, QRO2, QBRT3.1-3.2, QBRT8.2, and QRT9.1-9.2) [12, [17] [18] [19] . To analyze adaptation process of upland rice to aerobic conditions, a study based on 5779 single nucleotide polymorphisms (SNPs) was conducted and found that upland rice ecotypes have most robust roots (long and thick) and very high number of robust root alleles than other rice ecotypes [20] . SNP genotyping analyses between upland and lowland landraces found that two potentially drought-resistance genes (ARAG1 and OsGL1-8) underlying root architecture-associated drought avoidance may have undergone directional selection in upland rice [21] . Comparative transcriptome analysis similar to our approach was used to state the molecular mechanism of stress adaptation in upland rice, and ecotype differentiation genes were enriched in ROS alleviating gene families such as peroxidases, glutathione related classes and in phytohormone metabolism and signaling factors and transcription factors [22] [23] [24] . In flooded soils, roots develop in the shallow soil layer, which favors nutrient uptake from the floodwaters. In aerobic soils, by contrast, root growth is more dispersed [25] . AWD irrigation can increase root biomass, root length density, total root absorption area, active root absorption area and zeatin (Z) + zeatin riboside (ZR) content in roots when compared to those under CF irrigation which may contribute to higher grain yield and water productivity [26] . Rice shows greater root activity under AWD irrigation when compared to CF irrigation [27] . More developed root systems of upland rice helped the plants maintain a higher water status than that maintained by lowland rice when the plants were subjected to soil drying [28] . Phytohormones may play important regulatory role in crop growth by acting as signaling molecules to regulate crop physiological function and metabolism [29] . Accumulation of Abscisic acid (ABA) regulates auxin transport in the root tip, which enhances proton secretion (protons cause cell extension by cell wall loosening and then cause root growth) for maintaining root growth under moderate water stress [30] . Many transcription factors (TFs) are also reported to regulate root growth [31, 32] . However, regulation of these phytohormones and TFs during root growth in lowland and upland rice under AWD is yet to be understood fully. In recent times, utilization of transcriptome approaches such as microarrays and next-generation sequencing have helped researchers to fish out genome-wide gene expression changes. RNA-Seq uses deep-sequencing technology to provide a far more precise measurement of the levels of transcripts and their isoforms than other methods such as microarrays [33] . RNA-seq allows gene discovery and global gene expression profiling, for example, to identify key signalling components of pathogen-resistance pathways [34] . With the rapidly decreasing Journal Pre-proof potential was measured using tensiometers (two tensiometers in each AWD irrigated pot) at midday each day. According to the measurement, when the soil water potential reached −15 kPa under the AWD irrigation, water was applied to a depth of 2-3 cm in the corresponding AWD pots. The quantity of water applied was quantified based on the number of cups used to add water. In total, 15 and 9 irrigation events were applied before sampling date in CF and AWD irrigation, respectively. The heading date of Nipponbare under AWD and CF was between June 18-26. The heading date of Gaoshan 1 under AWD and CF was between June 20-28. Root dry weight (RDW) and root oxidation activity (ROA) Rice plants were sampled destructively from the 12 dedicated pots at heading stage (June 23), i.e. 3 replicates × 2 irrigation regimes × 2 varieties. Each pot contained three root samples which were collected to analyze RDW, ROA and transcriptome respectively. For RDW, all of the above-ground tissues were separated by cutting the rice plants at soil surface and roots were collected by gently removing the surrounding saturated soil and washing over a 2000 μm mesh size sieve. Samples were dried in an oven at 70 °C until constant weight, and then RDW was Journal Pre-proof Library examination, clustering and sequencing Library insert size was quantified using StepOnePlus Real-Time PCR System (Library valid concentration > 10 nmol L −1 ). Sample clustering was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina). Subsequently, libraries were sequenced on an Illumina platform to obtain 150 bp paired-end reads. Transcriptome analysis Reads were processed through quality check using FastQC protocol and high-quality reads (referred to as "clean reads") were obtained for downstream mapping to reference genome of rice (ftp://ftp.ensemblgenomes.org/ pub/plants/release-24/fasta/oryza_sativa/) in HISAT2 v2.0.5 [41] . Following this, raw read count for each gene in each sample was obtained with HTSeq v0.6.0, and FPKM (Fragments Per Kilobase Million mapped reads) was calculated to estimate the expression level of genes in each sample. Finally, DESeq2 v1.6.3 was used for Journal Pre-proof database was performed. The top 20 KEGG pathways involving most DEGs were listed (Fig. 5) . For Nipponbare, the DEGs were mostly enriched in the terms such as "Phenylpropanoid biosynthesis, Biosynthesis of amino acids, Carbon metabolism, Ribosome and Plant hormone signal transduction" (Fig. 5-A) ; whereas for Gaoshan 1, the Journal Pre-proof J o u r n a l P r e -p r o o f 9 DEGs were mostly enriched in "Protein processing in endoplasmic reticulum, Plant-pathogen interaction, Ubiquitin mediated proteolysis, Spliceosome and Phenylpropanoid biosynthesis" (Fig. 5-B) . For the two cultivars, the common enriched pathways were: Phenylpropanoid biosynthesis, Protein processing in endoplasmic reticulum, Carbon metabolism, Plant hormone signal transduction, Plant-pathogen interaction, Starch and sucrose metabolism, MAPK signaling pathway-plant, and Endocytosis. Journal Pre-proof Response of protein processing in ER pathway to AWD irrigation Proteins are modified and folded in the ER, and one-third of newly synthesized proteins are misfolded [53] . Misfolded proteins can be folded with the assistance of ER chaperones, or destroyed by ERAD [54]. Abiotic stress can induce the accumulation of unfolded proteins in the ER and cause ER stress [55] . ER response pathway was reported to regulate root growth when Arabidopsis were under water stress [56] . In our work, 24 and 6 genes involved in ERAD were differentially expressed in the roots of Nipponbare and Gaoshan 1 (Fig. 6) , respectively, and four common HSP20 family genes involved in ERAD were up-regulated in the roots of both rice varieties. Genes involved in ER stress recovery (EIF2AKs) were down-regulated in both rice varieties, and 4 genes involved in protein targeting (PDIAs) were specifically down-regulated in Nipponbare, while one gene involved in protein correctly folded (HUGT) was specifically down-regulated in Gaoshan 1. These showed that protein processing in ER pathway may be involved in the AWD irrigation adaption of rice roots. Response of lignin biosynthetic pathway to AWD irrigation