期刊：nature communications (17.694/Q1)
Natural variation ofDROT1confers drought adaptation in upland rice
DROT1的自然变异赋予旱稻干旱适应能力
Abstract
Upland rice is a distinct ecotype that grows in aerobic environments and tolerates drought stress. However, the genetic basis of its drought resistance is unclear. Here, using an integrative approach combining a genome-wide association study with analyses of introgression lines and transcriptomic profiles, we identify a gene, DROUGHT1 (DROT1), encoding a COBRA-like protein that confers drought resistance in rice. DROT1 is specifically expressed in vascular bundles and is directly repressed by ERF3 and activated by ERF71, both drought responsive transcription factors. DROT1 improves drought resistance by adjusting cell wall structure by increasing cellulose content and maintaining cellulose crystallinity. A C-to-T single-nucleotide variation in the promoter increases DROT1 expression and drought resistance in upland rice. The potential elite haplotype of DROT1 in upland rice could originate in wild rice (O. rufipogon) and may be beneficial for breeding upland rice varieties.
旱稻是一种独特的生态型，可在有氧环境中生长并耐受干旱胁迫。 然而，其抗旱性的遗传基础尚不清楚。 在这里，使用一种综合方法，将全基因组关联研究与基因渗入系和转录组谱分析相结合，我们确定了一个基因 DROUGHT1 (DROT1)，该基因编码一种赋予水稻抗旱性的 COBRA 样蛋白。 DROT1 在维管束中特异性表达，并被 ERF3 直接抑制并被 ERF71 激活，这两种转录因子都是干旱反应性转录因子。 DROT1 通过增加纤维素含量和维持纤维素结晶度来调整细胞壁结构，从而提高抗旱性。 启动子中的 C 到 T 单核苷酸变异增加了旱稻的 DROT1 表达和抗旱性。 旱稻 DROT1 的潜在优良单倍型可能起源于野生稻（O. rufipogon），可能有利于培育旱稻品种。
Drought is a persistent impediment to crop production, especially in the context of global climate change and growing water demand for agriculture. Rice is a staple food for more than half of the world’s population, but in China, for example, 70% of agricultural water is devoted to rice production alone. Availability of irrigation water will increasingly become a limiting factor for rice cultivation. Thus, breeding rice cultivars that require less water and have excellent drought resistance is an urgent necessity.
干旱是作物生产的长期障碍，尤其是在全球气候变化和农业用水需求不断增长的背景下。 大米是世界一半以上人口的主食，但以中国为例，仅大米生产就有 70% 的农业用水。 灌溉水的供应将日益成为水稻种植的限制因素。 因此，培育出耗水量少、抗旱性好的水稻品种势在必行。
Upland rice was domesticated in rain-fed regions and grows under aerobic soil conditions, whereas lowland rice is grown in paddy fifields with its basal section covered by water. There are obvious genetic differences between upland and lowland rice in regard to drought resistance and productivity-related traits. However, the genetic basis of aerobic drought-adaptive traits remains unclear, which greatly limits the potential utilization of gene variations in breeding. Linkage analyses have been performed using multiple drought-related traits to elucidate the genetic mechanism of drought resistance in rice. However, cloning genes in such ways is challenging because drought resistance is a complex agronomic trait that is regulated by multiple loci with minor effects. Recently, genome-wide association studies (GWASs) have been used to explore the genetics of drought resistance in maize and rice, and several drought-related genes have been functionally elucidated. However, further research is still needed to determine the genetic basis of drought related trait differentiations between upland and lowland rice.
旱稻在雨养地区驯化并在好氧土壤条件下生长，而低地稻则生长在稻田中，其基部被水覆盖。旱稻和低地稻在抗旱性和生产力相关性状方面存在明显的遗传差异。然而，好氧干旱适应性状的遗传基础仍不清楚，这极大地限制了基因变异在育种中的潜在利用。已经使用多种干旱相关性状进行了连锁分析，以阐明水稻抗旱性的遗传机制。然而，以这种方式克隆基因具有挑战性，因为抗旱性是一种复杂的农艺性状，受多个基因座调控，影响较小。最近，全基因组关联研究（GWASs）已被用于探索玉米和水稻抗旱性的遗传学，并且已经在功能上阐明了几个与干旱相关的基因。然而，仍需要进一步的研究来确定旱稻和低地稻之间干旱相关性状分化的遗传基础。
As the first line of defense against various abiotic and biotic environmental stresses, cell walls play important roles in plant resistance to adversity. Recent studies have revealed that mutations in genes affecting cell wall composition and/or structure can alter resistance to drought or other abiotic stresses in maize and rice. Plants can monitor their cell wall architecture under normal growth and changing environments. During normal plant cell growth, cell expansion is controlled by the directional organization of cellulose microfibrils in the cell wall, which is regulated by COBRA proteins. In rice, the COBRA family comprises 11 members, among which BC1 is crucial for cell wall formation; its functional mutation causes brittle culm due to a reduction in cell wall thickness and cellulose content. OsBC1L4, a homolog of BC1, is also associated with cell wall formation and plant height. Although the COBRA family genes are important for plant cell wall formation, their roles in responding to drought stress, balancing cell growth and resistance to abiotic stress are still unknown.
作为抵御各种非生物和生物环境胁迫的第一道防线，细胞壁在植物抵御逆境中发挥着重要作用。最近的研究表明，影响细胞壁组成和/或结构的基因突变可以改变玉米和水稻对干旱或其他非生物胁迫的抵抗力。植物可以在正常生长和变化的环境下监测它们的细胞壁结构。在正常的植物细胞生长过程中，细胞扩张受细胞壁中纤维素微纤维的定向组织控制，该组织受 COBRA 蛋白的调节。在水稻中，COBRA 家族共有 11 个成员，其中 BC1 对细胞壁形成至关重要；由于细胞壁厚度和纤维素含量的减少，其功能突变会导致茎秆变脆。 OsBC1L4 是 BC1 的同源物，也与细胞壁形成和植物高度有关。尽管 COBRA 家族基因对植物细胞壁的形成很重要，但它们在应对干旱胁迫、平衡细胞生长和抵抗非生物胁迫方面的作用仍然未知。
Drought resistance involves multiple physiological responses and is associated with the expression of numerous genes that are regulated by a complex transcriptional regulatory network. Among the transcription factors responding to drought stress, ERF family members are important regulators that act as transcriptional activators or repressors of GCC box-mediated gene expression. ERF3 is a transcriptional repressor, and its overexpression can reduce drought tolerance in rice, while overexpression of ERF3 with a mutated EAR motif has no significant effect on drought tolerance. Similarly, overexpression of OsAP2-39 (closely related to ERF3) can also inhibit the drought resistance of transgenic lines. In contrast, several other ERF genes act as positive regulators of drought resistance. For example, OsLG3 (OsERF62) increases drought tolerance by inducing ROS scavenging. ERF71 improves drought resistance by regulating the expression of genes involved in cell wall lignification. However, the ERF genes that regulate drought resistance by affecting cell wall structure are still poorly understood.
抗旱性涉及多种生理反应，并与受复杂转录调控网络调控的众多基因的表达有关。在响应干旱胁迫的转录因子中，ERF 家族成员是重要的调节因子，它们充当 GCC 盒介导的基因表达的转录激活因子或抑制因子。 ERF3是一种转录抑制因子，其过表达会降低水稻的耐旱性，而带有突变EAR基序的ERF3过表达对耐旱性没有显着影响。同样，OsAP2-39（与ERF3密切相关）的过表达也可以抑制转基因品系的抗旱性。相比之下，其他几个 ERF 基因充当抗旱性的正调节剂。例如，OsLG3 (OsERF62) 通过诱导 ROS 清除来提高耐旱性。 ERF71 通过调节参与细胞壁木质化的基因的表达来提高抗旱性。然而，通过影响细胞壁结构来调节抗旱性的 ERF 基因仍然知之甚少。
In this work, we conduct a GWAS on a group of natural accessions composed of both upland and lowland rice and clone a drought resistance gene, DROT1, that encodes a COBRA family protein. DROT1 enhances drought resistance by adjusting cell wall structure and is directly repressed by ERF3 and activated by ERF71, in a mechanism that may help control the balance between plant growth and drought resistance. Additionally, natural variation in the promoter of DROT1 confers divergent drought resistance on upland and lowland rice, showing potential value for breeding.
在这项工作中，我们对一组由旱稻和低地稻组成的自然种质进行了 GWAS，并克隆了编码 COBRA 家族蛋白的抗旱基因 DROT1。DROT1 通过调节细胞壁结构来增强抗旱性，并被 ERF3 直接抑制并被 ERF71 激活，这种机制可能有助于控制植物生长和抗旱性之间的平衡。此外，DROT1 启动子的自然变异赋予了旱稻和低地稻不同的抗旱性，显示出潜在的育种价值。
Results
Mining QTLs for drought resistance in japonica rice.
To investigate the genetic diversity of rice drought resistance, we evaluated the drought stress phenotypes of 271 rice germplasms including 59 upland rice and 212 lowland rice accessions under field drought conditions (Supplementary Data 1). The rolling degree and the color of seedling leaves are two important traits that showed obvious differences among rice accessions under drought stress (Supplementary Fig. 1a). We determined drought resistance of each germplasm by evaluating the Leaf Rolling Index (LRI) and Leaf Color Index (LCI) and then calculating the Drought Resistance Index (DRI) based on appropriate weighting of the LRI and LCI (see Methods). Under drought stress, varieties with different DRI values generally exhibited significantly different growth performances, indicating that the DRI can be used to reflflect the drought resistance of rice seedlings (Fig. 1a). The DRI values of upland rice accessions were significantly higher than those of lowland rice accessions, which is consistent with the better drought resistance of upland rice (Fig. 1b). The high phenotypic diversity and continuous variation of DRI and LRI indicated that these germplasms contained multiple drought resistant QTLs (Supplementary Fig. 1b, c). Principal component analysis (PCA) of single-nucleotide polymorphisms (SNPs) in linkage equilibrium showed that temperate japonica, tropical japonica and sub-tropical japonica accessions formed distinct clusters, indicating that the set of 271 germplasm represents a structured population (Supplementary Fig. 1d). Using 2,070,333 SNPs covering the entire rice genome, we performed a GWAS for drought resistance under a Compressed Mixed Linear Model (CMLM), which identified seven loci as significantly associated with DRI (Fig. 1c, Supplementary Fig. 2a, and Supplementary Data 2). High contribution rate of LRI to DRI (89.5%) and the significantly different LRI between upland and lowland rice subgroups indicate that LRI is the main component of DRI. Therefore, we performed a GWAS on LRI and identified seven loci that were significantly associated with LRI (Fig. 1d, Supplementary Fig. 2b, Supplementary Data 2). Among them, qDR4a, qDR4b and qDR10b also contributed to DRI.
为了研究水稻抗旱性的遗传多样性，我们评估了 271 种水稻种质的干旱胁迫表型，包括 59 种旱稻和 212 种低地水稻种质在田间干旱条件下（补充数据 1）。滚动程度和苗叶颜色是干旱胁迫下水稻种质之间表现出明显差异的两个重要性状（补充图1a）。我们通过评估叶滚动指数（LRI）和叶色指数（LCI），然后根据 LRI 和 LCI 的适当加权计算抗旱指数（DRI）来确定每种种质的抗旱性（见方法）。在干旱胁迫下，不同DRI值的品种通常表现出显着不同的生长性能，表明DRI可用于反映水稻幼苗的抗旱性（图1a）。旱稻种质的DRI值显着高于低地稻种质，这与旱稻更好的抗旱性一致（图1b）。 DRI和LRI的高表型多样性和连续变异表明这些种质含有多个抗旱QTL（补充图1b，c）。连锁平衡中单核苷酸多态性（SNP）的主成分分析（PCA）表明温带粳稻、热带粳稻和亚热带粳稻种质形成了不同的簇，表明这组271个种质代表了一个结构化的种群（补充图1d） ）。使用覆盖整个水稻基因组的 2,070,333 个 SNP，我们在压缩混合线性模型（CMLM）下进行了抗旱性 GWAS，该模型确定了七个与 DRI 显着相关的基因座（图 1c、补充图 2a 和补充数据 2） . LRI 对 DRI 的贡献率高（89.5%）以及旱稻和低地稻亚组之间 LRI 的显着差异表明 LRI 是 DRI 的主要组成部分。因此，我们对 LRI 进行了 GWAS 并确定了与 LRI 显着相关的七个基因座（图 1d，补充图 2b，补充数据 2）。其中，qDR4a、qDR4b和qDR10b对DRI也有贡献。
To further confirm the drought resistance loci, we simulated water-deficit stress on a set of 88 introgression lines (ILs) with segments from IRAT109 (an upland tropical japonica accession) in the background of Yuefu (a lowland temperate japonica accession). After these lines were germinated on 15% polyethylene glycol (PEG) 6000 (w/v) for 10 days, the relative shoot lengths of 32 ILs were significantly increased compared with that of Yuefu (P < 0.001) (Supplementary Data 3). Among these, IL349, carrying qDR10b, and IL10, carrying qDR4a and qDR4b, showed greater relative shoot length than Yuefu (Fig. 1e, f), which further confirmed that qDR4a, qDR4b, and qDR10b are important in rice drought resistance.
为了进一步确认抗旱基因座，我们在乐府（低地温带粳稻种质）背景下模拟了一组 88 条渐渗系（ILs）的缺水胁迫，其中 IRAT109（一种高地热带粳稻种质）的片段。 这些品系在 15% 聚乙二醇 (PEG) 6000 (w/v) 上萌发 10 天后，与悦府相比，32 个 ILs 的相对枝条长度显着增加（P < 0.001）（补充数据 3）。 其中，携带qDR10b的IL349和携带qDR4a和qDR4b的IL10显示出比悦府更大的相对茎长（图1e，f），这进一步证实了qDR4a、qDR4b和qDR10b在水稻抗旱中的重要作用。
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