Integrative Analysis of Transcriptomic and Physiological Reveals Drought Adaption Strategies in Different Maize Genotypes


 Background: Drought is an environmental stress that adversely affects maize productivity. However, drought adaption strategies of different maize varieties are not fully clear at the transcriptomic level. In the paper, drought-sensitive SD902 and -resistant SD609 varieties were analyzed to explore transcriptional and physiological alterations to drought stress. Results: The higher SOD, CAT, GSH enzymatic antioxidants, stomatal conductance, transpiration, net photosynthesis rate suggested better performance of SD609 than SD902 variety under drought stress. In transcriptome profiling, a total of 8985 and 7305 difference expression genes (DEGs) were identified in SD902 and SD609 respectively. These genes were overall involved in antioxidation reduce, osmotic adjustment, protein modification (e.g. HSP and chaperone protein), photosynthesis, phytohormone (e.g. ABA, IAA, ethylene), transcription factors (TFs) (e.g. ERF, WRKY, NAC and bZIP) and MAPK (MAPK1/8, MKK4/9 and MKKK17) cascade. Among them, the upregulated genes significantly correlated with stress adjustment, HSPs and chaperone functions might better reduce drought-induced damage in both SD902 and especially SD609. The higher genes expression of IAA, ethylene and electron transfer in SD609 may be closely related to drought-tolerant performance than SD902 plants. Moreover, the misregulation of TFs, MAPK and ABA signaling would appear vital to explain the various sensitivity to drought in both varieties. Conclusion: The more drought-tolerant SD609 presented a beneficial and significantly higher genes expression of stress protection, IAA transduction, photosynthesis compared with drought-sensitive SD902 variety. Our findings provide vital insights into the molecular signatures underpinning drought resistance in maize.

3 Background 38 Sufficient water supply is essential for land plants growth and reproduction in natural 39 environment. However, increasing global temperature leads to more frequency drought 40 risk in agricultural production [1][2][3]. Drought adversely affect photosynthesis and 41 thereby cause excessive accumulation of reactive oxygen species (ROS) that damage 42 the plant growth and survival by oxidation [4]. To cope with the damage under drought 43 stressful conditions, plants have evolved multiple strategies to adaption to drought 44 condition [5]. For example, plants appropriately close stomatal to reduce water loss; 45 decrease photoinhibition to protect photosystem; improve antioxidant level to decrease 46 oxidative damage; induce heat shock proteins (HSPs) and molecular chaperones to 47 protect proteins [6,7]. At the molecular levels, molecular sensors promote the signal 48 transduction and thereby activate various transcriptional regulators. Ultimately, the 49 upstream controls result in a great variety of activation of genes and proteins to achieve 50 stress adjustment and growth [7]. Also, phytohormones [8], transcription factors (TFs) 51 [9] and others drought-responsive factors also widely participate in the response to 52 drought in plant. In general, drought adaption process involved in multiple metabolism 53 pathways that cause complex regulation mechanism in plants. 54 With rapid development of high-throughput sequencing technology, transcriptomic 55 has provided huge amounts of transcriptional evidences to systematically compare and 56 analyze complex mechanism in response to drought stress in plants [10]. Currently, 57 transcriptomic analyses has been widely used to reveal biological adaption to drought  106 stress 107 Based on the RNA-seq, the transcription profiles of leaf samples from SD609 and 108 SD902 varieties exposed to drought and normal treatment condition then were analyzed 109 and compared systematically to study gene expression. Twelve cDNA libraries were DEGs (3892 up-regulated and 3413 down-regulated) in SD609 with the standards of 120 fold changes > 1 and P-value < 0.05 were found respectively ( Fig. 2A, Table S1). The

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Venn diagram discovered that 4707 DEGs were common to two drought stress 122 treatment groups, of which, 2413 DEGs were increased by drought pressure (Fig. 2B).

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These DEGs may play the important roles in the adaption to drought stress.  to photosystem I reaction center subunits were remarkably reduced by 1.97 to 6.10 179 times among SD902 and SD609. Drought stress also adversely affected expression of 180 Cyt b6/f complex (petB), plastocyanin (PC) and ferredoxin (Fd). Apart from four Fd 181 genes of SD902 and two Fds of SD609, other DEGs involved in electron transport 182 elements were drastically decreased in SD902 and SD609 such as petB (cyt b6), petE 183 (PC) and petF (Fd) genes. Accordingly, compared with SD902 variety, we also found 184 that SD609 variety had a higher effective quantum yield (Y(Ⅰ)), electron transport rate 185 (ETR(Ⅰ)), the quantum yield of regulatory energy dissipation (Y(NPQ)) and the 186 quantum yield of non-regulatory energy dissipation (Y(NO)) to drought (Fig. 3A, B, G 187 and H). Effective quantum yield (Y(II)) and the electron transport rate (ETR(II)) only 188 displayed slightly difference in SD902 and SD609 (Fig. 3E, F). The Y(NA) of SD902 189 had a higher level than SD609, while Y(ND) was significantly decreased in two Upon drought stress treatment, as shown in Table 3, the expression abundance of genes respectively, and the other 12 DEGs were decreased by 1.17 to 2.71 times. The analysis 208 of qRT-PCR and enzyme activity found that drought stress significantly decreased 209 enzyme activity and gene transcription abundance for PPDK, PEPC, NADP-ME and 210 Rubisco in SD902 and SD609 ( Fig. 3I-P). The NADP-ME and Rubisco had an obvious 211 difference in maize varieties after drought treatment, while genes expression level of 212 PEPC and rbcS displayed significant difference. Moreover, we also found many DEGs 213 were involved in glucose metabolism in SD902 and SD609 under drought-adaption 214 process (Table 4). More than 76% DEGs were downregulated impacting starch and 215 sucrose biosynthesis, such as starch synthase, granule-bound starch synthase (GBSS), 216 hexokinase (HK), sucrose-6-phosphatase and sucrose-phosphate synthase (SPS).

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Except for one alpha-amylase gene (LOC103651265) was upregulated, and more than 218 83% DEGs related to starch and sucrose degradation were slightly downregulated 219 including beta-amylase, beta-fructofuranosidase and sucrose synthase in SD902 and 220 SD609. Furthermore, 53% and 33% beta-glucosidase genes involved in cellulose 221 degradation were significantly upregulated in both SD902 and especially SD609. In  After drought stress treatment, ten DEGs related to osmotic adjustment were identified 228 in SD902 and SD609 varieties. As shown in Table S3, (Table S4). We compared the top ten TF families in two maize varieties  Furthermore, the interaction analysis of TFs in two varieties discovered that SD902 276 variety has a more complex biological relationship involved in more TF genes than 277 SD609 (Fig. 4B), which may imply a more efficient response prosses to drought by the 278 regulation of TFs. After drought stress treatment, plants phenotype from two maize hybrids displayed a 315 significant difference in leaves color and shape compared with well-watered treatment.

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The drought-sensitive SD902 have a weaker drought-tolerant performance (Fig. 1A).    (Table 1, 2). Accordingly, SD609 variety display a higher Y(Ⅰ), 360 ETR (Ⅰ) and Y (NPQ) than SD902, ultimate causing batter photosynthetic efficiency to 361 adaption drought (Fig. 1, 3). ATPase as main component on photosynthesis system SBPase were significantly downregulated (Fig. 3, Table 3). The enzymes activity of 367 carbon fixation in C4 plant photosynthesis determine the utilization rate of CO2 in the 368 intercellular space [28]. These evidences show that key genes involved in  Importantly, transcriptome data found significantly up-regulated polysaccharide 377 degradation genes, such as α-amylase, beta-fructofuranosidase (Table 4) (Table S3). The 403 major TFs members including ERF, WRKY, NAC and bZIP were significantly increased, 404 whereas the MYB and bHLH were decreased in drought stress condition (Fig. 4). 405 Previous research shown that overexpression of ERF genes in Arabidopsis, rice, tomato 406 and tobacco were able to enhance tolerance capability under diverse biotic and abiotic 407 stresses [47,48]. Here, more than 60% ERF TFs in SD902 and SD609 were upregulated  (Table 6). These plant hormones displayed a 433 response difference in quantity and type in two maize varieties exposed to drought  drought-induced factors regulation to cope better drought stress in SD609. Accordingly, 448 we proposed a molecular adaption network to drought based on two contrasting maize 449 (Fig. 5). The study is helpful to further explore drought-tolerant mechanisms and 450 develop cultivars withstand. SD609 seedings were exposed to drought stress condition (50 ± 5% SWC) for five days 465 by controlled water measure. Finally, the collected leaves samples with three biological 466 replicates for each treatment were frozen immediately in liquid nitrogen and stored at -Energy conversion efficiency measurement 478 According to the previous methods [21], The quantum yields of photosystem I (PSI)