Regulation of Root Exudation in Wheat Plants in Response to Alkali Stress
《鹼脅迫下小麥根系分泌物的調控機制》

Abstract  摘要

Soil alkalization is an important environmental factor limiting crop production. Despite the importance of root secretion in the response of plants to alkali stress, the regulatory mechanism is unclear. In this study, we applied a widely targeted metabolomics approach using a local MS/MS data library constructed with authentic standards to identify and quantify root exudates of wheat under salt and alkali stresses. The regulatory mechanism of root secretion in alkali-stressed wheat plants was analyzed by determining transcriptional and metabolic responses. Our primary focus was alkali stress-induced secreted metabolites (AISMs) that showed a higher secretion rate in alkali-stressed plants than in control and salt-stressed plants. This secretion was mainly induced by high-pH stress. We discovered 55 AISMs containing –COOH groups, including 23 fatty acids, 4 amino acids, 1 amino acid derivative, 7 dipeptides, 5 organic acids, 9 phenolic acids, and 6 others. In the roots, we also discovered 29 metabolites with higher levels under alkali stress than under control and salt stress conditions, including 2 fatty acids, 3 amino acid derivatives, 1 dipeptide, 2 organic acids, and 11 phenolic acids. These alkali stress-induced accumulated carboxylic acids may support continuous root secretion during the response of wheat plants to alkali stress. In the roots, RNAseq analysis indicated that 5 6-phosphofructokinase (glycolysis rate-limiting enzyme) genes, 16 key fatty acid synthesis genes, and 122 phenolic acid synthesis genes have higher expression levels under alkali stress than under control and salt stress conditions. We propose that the secretion of multiple types of metabolites with a –COOH group is an important pH regulation strategy for alkali-stressed wheat plants. Enhanced glycolysis, fatty acid synthesis, and phenolic acid synthesis will provide more energy and substrates for root secretion during the response of wheat to alkali stress.
土壤鹼化是限制作物生產的重要環境因子。儘管根系分泌物在植物應對鹼脅迫的反應中具有重要性，其調控機制仍不清楚。本研究採用廣泛靶向代謝組學方法，利用以標準品建構的本地 MS/MS 數據庫，對小麥在鹽分和鹼脅迫下的根系分泌物進行鑑定與定量分析。透過測定轉錄組和代謝組反應，我們分析了鹼脅迫小麥植株根系分泌的調控機制。我們主要關注鹼脅迫誘導分泌的代謝物（AISMs），這些代謝物在鹼脅迫植株中的分泌率高於對照組和鹽脅迫植株。此種分泌主要受高 pH 值脅迫誘導。我們發現了 55 種含有-COOH 基團的 AISMs，包括 23 種脂肪酸、4 種胺基酸、1 種胺基酸衍生物、7 種二肽、5 種有機酸、9 種酚酸以及 6 種其他化合物。 在根部，我們還發現了 29 種代謝物在鹼脅迫下的含量高於對照組和鹽脅迫條件，包括 2 種脂肪酸、3 種氨基酸衍生物、1 種二肽、2 種有機酸和 11 種酚酸。這些由鹼脅迫誘導累積的羧酸類物質，可能支持小麥植株在應對鹼脅迫期間持續進行根系分泌。根部 RNAseq 分析顯示，5 個 6-磷酸果糖激酶（糖解作用限速酶）基因、16 個關鍵脂肪酸合成基因以及 122 個酚酸合成基因，在鹼脅迫下的表現量均高於對照組和鹽脅迫條件。我們認為，分泌多種帶有-COOH 基團的代謝物是鹼脅迫小麥植株重要的 pH 調節策略。增強的糖酵解、脂肪酸合成和酚酸合成將為小麥應對鹼脅迫期間的根系分泌提供更多能量和底物。

1. Introduction  1. 緒論

As the ecological environment continues to deteriorate through unreasonable development and use, the global area of saline land has increased yearly [1,2,3,4,5]. The harmful salts in saline soils mainly include NaCl, Na₂SO₄, NaHCO₃, and Na₂CO₃. About 46% of saline soils contain only neutral salts, NaCl, and Na₂SO₄, but the remaining 54% contain both neutral salts and alkaline salts [6]. The stress type exerted by NaCl and/or Na₂SO₄ is defined as salt stress, whereas the stress type exerted by NaHCO₃ and/or Na₂CO₃ is defined as alkali stress [7,8]. Previous studies have verified that the destructive effect of alkaline salt stress on plants is significantly stronger than that of neutral salt stress at the same salinity [7,8,9]. Soil alkalization has caused serious environmental problems in some areas of the world. For example, in northeastern China, about 50% of grassland is threatened by soil alkalization [10]. Soil pH in the alkalized area even reaches above 10.5. Only a few alkali-resistant halophytes can survive under such heavily alkaline conditions, and no crop can survive extreme alkalinity. Therefore, further research on soil alkalization and alkali stress is warranted.
隨著不合理的開發利用導致生態環境持續惡化，全球鹽鹼地面積逐年增加[1, 2, 3, 4, 5]。鹽鹼土壤中的有害鹽類主要包括 NaCl、Na₂SO₄、NaHCO₃和 Na₂CO₃。約 46%的鹽鹼土壤僅含有中性鹽 NaCl 和 Na₂SO₄，其餘 54%則同時含有中性鹽與鹼性鹽[6]。由 NaCl 和/或 Na₂SO₄所產生的脅迫類型被定義為鹽脅迫，而由 NaHCO₃和/或 Na₂CO₃所產生的脅迫類型則定義為鹼脅迫[7,8]。先前研究證實，在相同鹽度條件下，鹼性鹽脅迫對植物的破壞效應顯著強於中性鹽脅迫[7, 8, 9]。土壤鹼化已在全球部分地區造成嚴重的環境問題，例如中國東北地區約有 50%的草原正受到土壤鹼化威脅[10]。鹼化區域的土壤 pH 值甚至高達 10.5 以上，在此類強鹼性環境下僅有少數耐鹼鹽生植物能夠存活，一般作物皆無法在極端鹼性條件下生存。 因此，針對土壤鹼化與鹼脅迫的進一步研究是必要的。

Salt stress produces negative effects on plants through osmotic stress and ion toxicity. However, in addition to osmotic stress and ion toxicity, alkali stress produces high-pH stress. High pH caused by alkali stress can lead to the precipitation of Ca²⁺, Mg²⁺, Fe²⁺, Mn²⁺, Cu²⁺, Zn²⁺, and PO₄³⁻ to surrounding roots, which induces a reduction in the bioavailability of nutrient elements [2,9]. Additionally, a proton gradient across root plasma membranes is the driving force for mineral ion uptake. HCO₃⁻ or CO₃²⁻ from alkaline soils will neutralize the proton outside the root plasma membrane, thus breaking the proton gradient and inhibiting the uptake of mineral ions. The plants living in alkaline soils must regulate rhizosphere pH to alleviate nutrient stress. Therefore, the pH regulation of the roots is essential for alkali tolerance in plants.
鹽脅迫通過滲透脅迫和離子毒性對植物產生負面影響。然而，除了滲透脅迫和離子毒性外，鹼脅迫還會產生高 pH 值脅迫。鹼脅迫引起的高 pH 值會導致 Ca ²⁺ 、Mg ²⁺ 、Fe ²⁺ 、Mn ²⁺ 、Cu ²⁺ 、Zn ²⁺ 和 PO ₄ ³⁻ 在根系周圍沉澱，從而降低營養元素的有效性[2,9]。此外，根細胞膜兩側的質子梯度是礦質離子吸收的驅動力。來自鹼性土壤的 HCO ₃ ⁻ 或 CO ₃ ²⁻ 會中和根細胞膜外的質子，從而破壞質子梯度並抑制礦質離子的吸收。生長在鹼性土壤中的植物必須調節根際 pH 值以緩解營養脅迫。因此，根系的 pH 值調節對於植物的耐鹼性至關重要。

In the past 30 years, great progress has been made in several areas of salt stress study, such as ion homeostasis, signal transduction, and hormone regulation [11,12,13,14,15]. To date, multilevel signal networks mediating salt tolerance and Na⁺ compartmentalization mechanisms at the subcellular level have been elucidated [11,12,13,14,15,16]. However, relatively few studies have focused on plant alkali tolerance [3,4,17,18,19,20,21,22,23,24,25,26,27,28]. Important progress in research on plant alkali tolerance has been made in Arabidopsis [21], maize [25], and wheat [27], in which H⁺-ATPase was demonstrated to play an important role in alkali tolerance.
過去 30 年間，鹽分脅迫研究在離子平衡、信號傳導與激素調控等領域已取得重大進展[11,12,13,14,15]。迄今為止，學界已闡明介導耐鹽性的多層級信號網絡及亞細胞層面的鈉離子區隔化機制[11,12,13,14,15,16]。然而針對植物耐鹼性的研究相對匱乏[3,4,17,18,19,20,21,22,23,24,25,26,27,28]。目前關於擬南芥[21]、玉米[25]和小麥[27]的耐鹼性研究已取得重要進展，其中氫離子幫浦 ATP 酶被證實在耐鹼性中扮演關鍵角色。

Our group and other researchers have found that root secretion is the main pH regulation pathway of plants under alkali stress [29,30,31]. Root exudates usually include amino acids, phenolics, fatty acids, organic acids, and carbohydrates [22,32]. Secretion of organic acids induced by alkali stress has been reported in many plants, such as P. tenuiflora [30,33], grape plants [31], and Chloris virgata [29]. However, the physiological and molecular mechanisms underlying root secretion regulation during the response of plants to alkali stress are poorly understood. Wheat provides about 20% of the calories consumed by humans [34]. Soil alkalization is an important factor limiting wheat production in northern China. To explore the specific effects of high pH caused by alkali stress on root secretion, we applied salt stress and alkali stress treatments at the same Na⁺ concentration and total salt concentration but with different pH values. Thus, differences in plant root secretion in response to the two stress conditions were mainly attributed to pH differences. In this study, we identified and quantified root exudates of wheat under salt and alkali stresses. To ascertain the regulatory mechanism of root secretion in wheat under alkali stress, we also analyzed the transcriptional and metabolic responses of wheat roots to alkali stress.
我們的研究團隊及其他學者已發現，根部分泌作用是植物在鹼脅迫下調節 pH 值的主要途徑[29,30,31]。根系分泌物通常包含胺基酸、酚類物質、脂肪酸、有機酸及碳水化合物[22,32]。許多植物如羊草[30,33]、葡萄植株[31]及虎尾草[29]均被報導會在鹼脅迫下誘導有機酸分泌。然而，關於植物響應鹼脅迫時調控根部分泌作用的生理與分子機制仍所知甚少。小麥提供了人類攝取熱量的約 20%[34]，而土壤鹼化是限制中國北方小麥生產的重要因素。為探究鹼脅迫所致高 pH 值對根部分泌的具體影響，我們在相同 Na+濃度與總鹽濃度但不同 pH 值條件下，分別施加鹽脅迫與鹼脅迫處理，因此兩種脅迫條件下植物根部分泌的差異主要可歸因於 pH 值的不同。本研究針對小麥在鹽脅迫與鹼脅迫下的根系分泌物進行了鑑定與定量分析。 為釐清小麥在鹼脅迫下根部分泌的調控機制，我們同時分析了小麥根部對鹼脅迫的轉錄組與代謝組反應。

2. Results  2. 結果

2.1. Components of Root Exudates
2.1. 根部分泌物的組成成分

We used a high throughput metabolomic method to detect metabolites in the root exudates (Figure 1A) and root tissues of wheat plants (Figure 1B). Collectively, we detected 443 root exudates in wheat plants under three conditions (Figure 1A), including 75 fatty acids, 52 lipids, 27 organic acids, 31 amino acids or amino acid derivatives, 81 phenolic acids, 28 nucleotides or nucleotide derivatives, 54 flavonoids, 38 alkaloids, 7 terpenoids, 18 carbohydrates, 8 vitamins, 7 lignans or coumarins, and 17 others (Table S1). In wheat plants, 326 root exudates were detected under control conditions, 437 under salt stress, and 431 under alkali stress (Table S1 and Figure 1A). We particularly focused on alkali stress-induced secreted metabolites (AISMs), which were found at a higher root secretion rate under alkali stress condition than under control and salt stress conditions. The number of AISMs for each type of metabolite is displayed in Figure 2A,B. In Figure 2A, salt stress did not affect the secretion rate of the metabolites, but alkali stress enhanced the secretion rate. Conversely, in Figure 2B, both salt stress and alkali stress enhanced the secretion rate of the metabolites, with greater enhancement in alkali stress than in salt stress. We discovered 105 AISMs in wheat root exudates, including 27 fatty acids, 6 amino acids, 1 amino acid derivative, 7 dipeptides, 5 organic acids, 19 phenolic acids, 9 nucleotides or nucleotide derivatives, 6 flavonoids, 1 lignan or coumarin, 11 alkaloids, 2 carbohydrates, 1 terpenoid, 6 lipids, and 4 vitamins (Figure 1C and Figure 2). Of 105 AISMs, 55 AISMs contained the –COOH group, including 23 fatty acids, 4 amino acids, 1 amino acid derivative, 7 dipeptides, 5 organic acids, 9 phenolic acids, 3 alkaloids, 1 terpenoid, and 2 others (Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7). These data revealed that fatty acids, amino acids, dipeptides, and phenolic acids were dominant AISMs for alkali-stressed wheat plants. Some plant “hub” fatty acids, such as γ-linolenic acid, arachidonic acid, α-linolenic acid, linoleic acid, and palmitoleic acid also showed higher root secretion rates under alkali stress conditions than under control and salt stress conditions (Figure 4 and Figure 5). All of the three aromatic amino acids (tryptophan, tyrosine, and phenylalanine) were discovered in the list of AISMs (Figure 3).
我們採用高通量代謝組學方法檢測了小麥植株根系分泌物（圖 1A）和根組織中的代謝物（圖 1B）。總計在三種處理條件下共檢測到 443 種根系分泌物（圖 1A），包括 75 種脂肪酸、52 種脂質、27 種有機酸、31 種氨基酸或其衍生物、81 種酚酸、28 種核苷酸或其衍生物、54 種黃酮類化合物、38 種生物鹼、7 種萜類化合物、18 種碳水化合物、8 種維生素、7 種木脂素或香豆素，以及 17 種其他代謝物（表 S1）。小麥植株在對照條件下檢測到 326 種根系分泌物，鹽脅迫下 437 種，鹼脅迫下 431 種（表 S1 與圖 1A）。我們特別關注鹼脅迫誘導分泌代謝物（AISMs），這類代謝物在鹼脅迫條件下的根系分泌速率顯著高於對照組與鹽脅迫組。各類代謝物中 AISMs 的數量分布如圖 2A、B 所示。圖 2A 顯示鹽脅迫未影響代謝物分泌速率，而鹼脅迫則顯著提升了分泌速率。 相反地，在圖 2B 中，鹽脅迫和鹼脅迫都提高了代謝物的分泌速率，且鹼脅迫的增強效果大於鹽脅迫。我們在小麥根系分泌物中發現了 105 種 AISM（鹼脅迫誘導代謝物），包括 27 種脂肪酸、6 種胺基酸、1 種胺基酸衍生物、7 種二肽、5 種有機酸、19 種酚酸、9 種核苷酸或核苷酸衍生物、6 種黃酮類、1 種木脂素或香豆素、11 種生物鹼、2 種碳水化合物、1 種萜類、6 種脂質和 4 種維生素（圖 1C 和圖 2）。在這 105 種 AISM 中，有 55 種含有-COOH 官能基，包括 23 種脂肪酸、4 種胺基酸、1 種胺基酸衍生物、7 種二肽、5 種有機酸、9 種酚酸、3 種生物鹼、1 種萜類和 2 種其他物質（圖 3、圖 4、圖 5、圖 6 和圖 7）。這些數據顯示脂肪酸、胺基酸、二肽和酚酸是鹼脅迫小麥植株的主要 AISM。某些植物「樞紐」脂肪酸，如γ-亞麻酸、花生四烯酸、α-亞麻酸、亞油酸和棕櫚油酸，在鹼脅迫條件下的根系分泌速率也高於對照組和鹽脅迫條件（圖 4 和圖 5）。 三種芳香族胺基酸（色胺酸、酪胺酸與苯丙胺酸）均被發現列於 AISMs 清單中（圖 3）。

Figure 1. Comparison of metabolite components in root exudates and roots of wheat plants under control, salt stress, and alkali stress conditions. (A) Number of all detected root exudates; (B) number of all detected metabolites in roots; (C) number of the metabolites with enhanced root secretion rate; (D) number of the metabolites with upregulated accumulation in roots. The 30-day-old wheat seedlings were treated with salt (88 mM Na⁺ and pH 6.7) and alkali (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment.
圖 1. 小麥植株在對照組、鹽脅迫與鹼脅迫條件下根部滲出物與根部代謝物成分比較。(A) 所有檢測到的根部滲出物數量；(B) 所有檢測到的根部代謝物數量；(C) 根部分泌速率增強之代謝物數量；(D) 根部累積量上調之代謝物數量。30 日齡小麥幼苗分別以鹽溶液（88 mM Na ⁺ ，pH 6.7）與鹼溶液（88 mM Na ⁺ ，pH 8.8）處理 3 天。每種處理設置三個生物重複。

Figure 2. Alkali stress-induced secreted metabolites. The number of metabolites for each type of metabolite was displayed. (A) Control and salt-stressed plants showed a similar secretion rate for each metabolite, with a lower secretion rate than that in alkali-stressed plants; (B) alkali-stressed plants > salt-stressed plants > control plants in the secretion rate of metabolites. The 30-day-old wheat seedlings were treated with salt (88 mM Na⁺ and pH 6.7) and alkali (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment.
圖 2. 鹼脅迫誘導的分泌代謝物。顯示各類代謝物的數量。(A)對照組與鹽脅迫植株對各代謝物的分泌速率相近，但均低於鹼脅迫植株；(B)代謝物分泌速率呈現：鹼脅迫植株＞鹽脅迫植株＞對照組植株。試驗採用 30 天齡小麥幼苗，分別以鹽溶液(88 mM Na ⁺ ，pH 6.7)與鹼溶液(88 mM Na ⁺ ，pH 8.8)處理 3 天，每種處理設置 3 個生物重複。

Figure 3. Comparative effects of salt and alkali stresses on the secretion of amino acids and amino acid derivatives in wheat plants. Alkali stress-induced secreted amino acids or amino acid derivatives are displayed. The 30-day-old wheat seedlings were treated with salt stress (88 mM Na⁺ and pH 6.7) and alkali stress (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment. Different letters above the bar indicate significant differences.
圖 3. 鹽鹼脅迫對小麥植株分泌胺基酸及其衍生物的比較效應。圖中顯示鹼脅迫誘導分泌的胺基酸或胺基酸衍生物。30 天齡小麥幼苗分別接受鹽脅迫（88 mM Na ⁺ ，pH 6.7）與鹼脅迫（88 mM Na ⁺ ，pH 8.8）溶液處理 3 天。每組處理設置三個生物重複。柱狀圖上方不同字母表示顯著性差異。

Figure 4. Comparative effects of salt and alkali stresses on the secretion of unsaturated fatty acids in wheat plants. Alkali stress-induced secreted unsaturated fatty acids are displayed. The 30-day-old wheat seedlings were treated with salt (88 mM Na⁺ and pH 6.7) and alkali (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment. Different letters above the bar indicate significant differences.
圖 4. 鹽鹼脅迫對小麥植株分泌不飽和脂肪酸的比較效應。圖中顯示鹼脅迫誘導分泌的不飽和脂肪酸。30 天齡小麥幼苗分別接受鹽（88 mM Na ⁺ ，pH 6.7）與鹼（88 mM Na ⁺ ，pH 8.8）溶液處理 3 天。每組處理設置三個生物重複。柱狀圖上方不同字母表示顯著性差異。

Figure 5. Comparative effects of salt and alkali stresses on the secretion of saturated fatty acids in wheat plants. Alkali stress-induced secreted saturated fatty acids are displayed. The 30-day-old wheat seedlings were treated with salt stress (88 mM Na⁺ and pH 6.7) and alkali stress (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment. Different letters above the bar indicate significant differences.
圖 5. 鹽與鹼脅迫對小麥植株飽和脂肪酸分泌的比較效應。顯示鹼脅迫誘導分泌的飽和脂肪酸。30 天齡小麥幼苗分別以鹽脅迫溶液(88 mM Na ⁺ ，pH 6.7)與鹼脅迫溶液(88 mM Na ⁺ ，pH 8.8)處理 3 天。每種處理設置三個生物重複。柱狀圖上方不同字母表示顯著性差異。

Figure 6. Comparative effects of salt and alkali stresses on the secretion of phenolic acids with a –COOH group in wheat plants. Alkali stress-induced secreted phenolic acids are displayed. The 30-day-old wheat seedlings were treated with salt stress (88 mM Na⁺ and pH 6.7) and alkali stress (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment. Different letters above the bar indicate significant differences.
圖 6. 鹽與鹼脅迫對小麥植株含-COOH 基團酚酸分泌的比較效應。顯示鹼脅迫誘導分泌的酚酸。30 天齡小麥幼苗分別以鹽脅迫溶液(88 mM Na ⁺ ，pH 6.7)與鹼脅迫溶液(88 mM Na ⁺ ，pH 8.8)處理 3 天。每種處理設置三個生物重複。柱狀圖上方不同字母表示顯著性差異。

Figure 7. Comparative effects of salt and alkali stresses on the secretion of other carboxylic acids in wheat plants. The 30-day-old wheat seedlings were treated with salt stress (88 mM Na⁺ and pH 6.7) and alkali stress (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment. Different letters above the bar indicate significant differences.
圖 7. 鹽鹼脅迫對小麥植株其他羧酸分泌的比較影響。將 30 天齡的小麥幼苗分別以鹽脅迫（88 mM Na ⁺ ，pH 6.7）和鹼脅迫（88 mM Na ⁺ ，pH 8.8）溶液處理 3 天。每種處理設置三個生物重複。柱狀圖上方不同字母表示顯著性差異。

2.2. Metabolic Profiling of the Roots
2.2. 根部代謝輪廓分析

In wheat roots, we collectively detected 1011 metabolites, including 91 fatty acids, 93 lipids, 81 organic acids, 97 amino acids or amino acid derivatives, 164 phenolic acids, 71 nucleotides or nucleotide derivatives, 128 flavonoids, 111 alkaloids, 22 terpenoids, 64 carbohydrates, 16 vitamins, 44 lignans or coumarins, 5 quinones, and 24 others (Figure 1B,D and Table S2). Of these metabolites, 106 metabolites displayed different concentrations under control and salt stress conditions, 224 metabolites displayed different concentrations under control and alkali stress conditions, and 144 metabolites were differentially accumulated under salt stress and alkali stress conditions. We displayed alkali stress-induced accumulated metabolites (AIAMs), which were found at a higher concentration in the roots under alkali stress conditions than under control and salt stress conditions (Figure 8). The number of AIAMs for each type of metabolite is shown in Figure 8A,B. In Figure 8A, salt stress did not affect the accumulation of the metabolites, but alkali stress enhanced the accumulation. In Figure 8B, both salt stress and alkali stress enhanced the concentration of the metabolites, with greater enhancement in alkali stress than in salt stress. We discovered 29 AIAMs in wheat roots, including 2 fatty acids (γ-linolenic acid and α-linolenic acid), 3 amino acid derivatives, 1 dipeptide, 2 organic acids (shikimic acid and muconic acid), 11 phenolic acids, 2 flavonoids, 1 lipid, 1 terpenoid, and 6 alkaloids (Figure 8 and Table S3). Integrated analysis of root exudates and root metabolome data showed higher levels of γ-linolenic acid and α-linolenic acid in alkali-stressed roots than in control and salt-stressed roots, as well as a faster secretion rate in alkali-stressed roots than in control and salt-stressed roots.
在小麥根部，我們共檢測到 1011 種代謝物，包括 91 種脂肪酸、93 種脂質、81 種有機酸、97 種胺基酸或胺基酸衍生物、164 種酚酸、71 種核苷酸或核苷酸衍生物、128 種黃酮類化合物、111 種生物鹼、22 種萜類化合物、64 種碳水化合物、16 種維生素、44 種木脂素或香豆素、5 種醌類以及 24 種其他代謝物（圖 1B、D 及表 S2）。其中，106 種代謝物在對照組與鹽脅迫條件下呈現濃度差異，224 種代謝物在對照組與鹼脅迫條件下顯示不同濃度，另有 144 種代謝物在鹽脅迫與鹼脅迫條件間存在差異性累積。我們特別標示出鹼脅迫誘導累積代謝物（AIAMs），這類代謝物在鹼脅迫條件下的根部濃度顯著高於對照組與鹽脅迫條件（圖 8）。各類代謝物中 AIAMs 的數量分布如圖 8A、B 所示。圖 8A 顯示，鹽脅迫未影響代謝物累積，但鹼脅迫顯著促進其累積。 在圖 8B 中，鹽脅迫和鹼脅迫均提高了代謝物濃度，且鹼脅迫的增強效果大於鹽脅迫。我們在小麥根部發現了 29 種 AIAMs（逆境誘導代謝物），包括 2 種脂肪酸（γ-亞麻酸和α-亞麻酸）、3 種氨基酸衍生物、1 種二肽、2 種有機酸（莽草酸和黏康酸）、11 種酚酸、2 種黃酮類化合物、1 種脂質、1 種萜類化合物以及 6 種生物鹼（圖 8 和表 S3）。根部滲出物與根部代謝體數據的整合分析顯示，鹼脅迫根部中的γ-亞麻酸和α-亞麻酸含量高於對照組和鹽脅迫根部，且其分泌速率也快於對照組和鹽脅迫根部。

Figure 8. Alkali stress-induced accumulated metabolites in wheat roots. The number of metabolites in each type is displayed. (A) Control and salt-stressed plants showed similar levels for each metabolite, with lower levels than those in alkali-stressed plants; (B) alkali-stressed plants > salt-stressed plants > control plants in levels of metabolites. The 30-day-old wheat seedlings were treated with salt stress (88 mM Na⁺ and pH 6.7) and alkali stress (88 mM Na⁺ and pH 8.8) solutions for 3 days. Three biological replicates were used for each treatment.
圖 8. 小麥根部在鹼脅迫下累積的代謝產物。各類代謝產物數量如圖所示。(A)對照組與鹽脅迫處理植株各代謝產物含量相近，均低於鹼脅迫處理植株；(B)代謝產物含量呈現鹼脅迫處理植株＞鹽脅迫處理植株＞對照組植株。試驗採用 30 日齡小麥幼苗，分別以鹽脅迫溶液(88 mM Na ⁺ ，pH 6.7)與鹼脅迫溶液(88 mM Na ⁺ ，pH 8.8)處理 3 天，每種處理設置 3 個生物學重複。

2.3. Gene Expression Response in the Roots
2.3. 根部基因表達反應

The results of the RNAseq were validated with real-time quantitative PCR (qRT-PCR) (Table S4). In 10 of the 12 randomly selected genes, the fold changes of the RNAseq experiment were similar to those of the qRT-PCR experiment, indicating that the results of the RNAseq experiment were reliable (Table S4). Compared with the control, salt stress upregulated the expression of 2108 genes and downregulated the expression of 1470 genes, whereas alkali stress upregulated the expression of 8542 genes and downregulated the expression of 6764 genes. The expression level of 5967 genes was higher in alkali-stressed roots than in salt-stressed roots, and 8147 genes displayed a lower level of expression in alkali-stressed roots than in salt-stressed roots. Alkali stress-induced genes (AIGs) were considered those with an expression level higher in alkali-stressed plants than in control and salt-stressed plants. We discovered 5764 AIGs, which were exposed to KEGG enrichment. The AIGs were enriched in phenylpropanoid biosynthesis, amino acid metabolism, nitrogen metabolism, amino acid-related enzymes, phenylalanine metabolism, flavonoid biosynthesis, alpha-linolenic acid metabolism, and other pathways (Table S5). AIGs involved in alkali tolerance are shown in Figures S1–S6. The AIGs included 18 NRT1/PTR FAMILY (NPF) genes, 22 NRT genes (Figure S1), 11 1-aminocyclopropane-1-carboxylate (ACC) oxidase genes, and 38 ethylene-responsive transcription factor genes (Figure S2). In the AIG list, we also discovered 29 glycolysis/gluconeogenesis genes including 5 glycolysis rate-limiting enzyme (6-phosphofructokinase) genes, and 16 key fatty acid synthesis genes (4 FabG genes, 1 FabF gene, 1 medium-chain acyl-[acyl-carrier-protein] hydrolase gene and 4 long-chain acyl-CoA synthetase genes) (Figure S3). Additionally, we also found 122 phenolic acid synthesis genes in the list of AIGs (Table S5), including 4 phenylalanine ammonia-lyase (PAL, phenolic acid synthesis rate-limiting enzyme) genes, 7 4-coumarate-CoA ligase (4CL) genes, and 2 trans-cinnamate 4-monooxygenase genes (Figure S4). The expression level of 22 peptide transporter genes, 3 oligopeptide transporter genes, 6 protease genes, 1 ubiquitin-conjugating enzyme gene, and 13 E3 ubiquitin-protein ligase genes was also higher in alkali-stressed roots than in control and salt-stressed roots (Figures S5 and S6).
RNA 定序(RNAseq)的結果透過即時定量聚合酶鏈鎖反應(qRT-PCR)進行驗證(表 S4)。在隨機選取的 12 個基因中，有 10 個基因的 RNAseq 實驗倍數變化與 qRT-PCR 實驗結果相似，表明 RNAseq 實驗結果可靠(表 S4)。與對照組相比，鹽脅迫上調了 2108 個基因的表達，下調了 1470 個基因的表達；而鹼脅迫則上調了 8542 個基因的表達，下調了 6764 個基因的表達。有 5967 個基因在鹼脅迫根部的表達水平高於鹽脅迫根部，另有 8147 個基因在鹼脅迫根部的表達水平低於鹽脅迫根部。我們將在鹼脅迫植株中表達水平高於對照組和鹽脅迫植株的基因定義為鹼脅迫誘導基因(AIGs)。共發現 5764 個 AIGs，並對其進行了 KEGG 富集分析。 AIGs 在苯丙素生物合成、胺基酸代謝、氮代謝、胺基酸相關酶、苯丙胺酸代謝、類黃酮生物合成、α-亞麻酸代謝等途徑中顯著富集（表 S5）。參與鹼耐受性的 AIGs 如圖 S1-S6 所示。這些 AIGs 包含 18 個 NRT1/PTR 家族(NPF)基因、22 個 NRT 基因（圖 S1）、11 個 1-氨基環丙烷-1-羧酸(ACC)氧化酶基因，以及 38 個乙烯響應轉錄因子基因（圖 S2）。在 AIG 列表中，我們還發現了 29 個糖酵解/糖異生基因，其中包括 5 個糖酵解限速酶（6-磷酸果糖激酶）基因，以及 16 個關鍵脂肪酸合成基因（4 個 FabG 基因、1 個 FabF 基因、1 個中鏈醯基-[醯基載體蛋白]水解酶基因和 4 個長鏈醯基輔酶 A 合成酶基因）（圖 S3）。此外，我們還在 AIG 列表中發現 122 個酚酸合成基因（表 S5），包括 4 個苯丙胺酸解氨酶(PAL，酚酸合成限速酶)基因、7 個 4-香豆酸輔酶 A 連接酶(4CL)基因，以及 2 個反式肉桂酸 4-單加氧酶基因（圖 S4）。 在鹼脅迫處理的根部中，22 個肽轉運蛋白基因、3 個寡肽轉運蛋白基因、6 個蛋白酶基因、1 個泛素結合酶基因以及 13 個 E3 泛素蛋白連接酶基因的表現量，均高於對照組與鹽脅迫處理的根部（圖 S5 與 S6）。

3. Discussion  3. 討論

Root secretion has a vital role in the tolerance of plants to abiotic stresses, such as phosphorus deficiency, heavy metal pollution, aluminum toxicity, and alkali stress [22,32]. The roles of organic acid secretion in pH regulation under alkali stress have been reported in grapevine roots [35], grape plants [31], and C. virgata plants [29]. High pH caused by alkali stress can precipitate various mineral element ions at the rhizosphere, leading to nutrient deficiency [28]. High pH can also induce the over-accumulation of Na⁺ and enhance ion toxicity [28]. Thus, the regulation of pH at the rhizosphere or within roots is vital for plant survival under high alkali conditions. In this study, we detected a diverse array of metabolites covering most types of metabolites in the root exudates of alkali-stressed wheat plants. We particularly focused on secreted metabolites induced by alkali stress (high-pH). We discovered 55 AISMs contained a –COOH group, including 23 fatty acids, 4 amino acids, 1 amino acid derivative, 7 dipeptides, 5 organic acids, 9 phenolic acids, 3 alkaloids, 1 terpenoid, and 2 others. We propose that the secretion of multiple types of metabolites with the –COOH group may be an important pH regulation strategy for wheat roots under alkali stress. Recently, root exudates of a halophyte Puccinellia tenuiflora under alkali stress were also analyzed by a metabolomics approach [33]. In P. tenuiflora plants, 75 AISMs with the –COOH group were discovered, including 42 fatty acids, 3 amino acid derivatives, 22 phenolic acids, and 8 organic acids [33]. Our recently published work revealed that halophyte Leymus chinensis responded to alkali stress via the secretion of phenolic acids, free fatty acids, organic acids, and amino acids [36]. However, that study did not apply salt stress treatment, so the root secretion response of L. chinensis to a high pH was not explored. The above data demonstrated that the secretion of fatty acids, phenolic acids, and organic acids was the common response of plants to alkali stress. However, amino acids and dipeptides were discovered in AISMs of wheat and not in P. tenuiflora. This suggests that wheat and the halophyte P. tenuiflora have different pH regulation strategies under alkali stress. The secretion of amino acids and dipeptides may play more important roles in wheat alkali tolerance.
根系分泌物在植物對非生物脅迫（如磷缺乏、重金屬污染、鋁毒性和鹼脅迫）的耐受性中具有關鍵作用[22, 32]。有機酸分泌在鹼脅迫下調節 pH 值的作用已在葡萄根系[35]、葡萄植株[31]和虎尾草[29]中被報導。鹼脅迫引起的高 pH 值會使根際各種礦質元素離子沉澱，導致營養缺乏[28]。高 pH 值還會誘導 Na+過量累積並加劇離子毒性[28]。因此，調節根際或根內 pH 值對於植物在高鹼環境下的生存至關重要。本研究在受鹼脅迫小麥植株的根系分泌物中檢測到涵蓋多數代謝物類型的多樣化代謝物。我們特別關注由鹼脅迫（高 pH 值）誘導分泌的代謝物，發現 55 種鹼脅迫誘導分泌代謝物含有-COOH 官能基，包括 23 種脂肪酸、4 種胺基酸、1 種胺基酸衍生物、7 種二肽、5 種有機酸、9 種酚酸、3 種生物鹼、1 種萜類化合物和 2 種其他代謝物。 我們提出，分泌多種帶有–COOH 基團的代謝產物可能是小麥根系在鹼性脅迫下調節 pH 值的重要策略。近期研究同樣透過代謝體學方法分析了鹽生植物星星草（Puccinellia tenuiflora）在鹼脅迫下的根系分泌物[33]。在星星草植株中，共發現 75 種帶有–COOH 基團的鹼誘導分泌代謝物（AISMs），包括 42 種脂肪酸、3 種胺基酸衍生物、22 種酚酸及 8 種有機酸[33]。我們近期發表的研究亦揭示，鹽生植物羊草（Leymus chinensis）透過分泌酚酸、游離脂肪酸、有機酸及胺基酸來應對鹼脅迫[36]。然而該研究未施加鹽分脅迫處理，故未探討羊草根系對高 pH 值的分泌反應。上述數據表明，分泌脂肪酸、酚酸與有機酸是植物應對鹼脅迫的共通反應。值得注意的是，小麥的 AISMs 中發現的胺基酸與二肽並未出現於星星草，此差異暗示小麥與鹽生植物星星草在鹼脅迫下可能採取不同的 pH 調控策略。 胺基酸和二肽的分泌可能在小麥耐鹼性中扮演更重要的角色。

Glycolysis provides the reducing power (ATP and NADH) and carbon source for metabolisms and the root secretion process. In the wheat roots, five 6-phosphofructokinase (glycolysis rate-limiting enzyme) genes displayed higher expression levels under alkali stress than under control and salt stress conditions (Figure S3). Enhanced glycolysis will provide more reducing power and carbon sources for the synthesis of fatty acids, phenolic acids, and organic acids to support their secretion into the rhizosphere during the response of wheat to alkali stress. We also focused on metabolites with a higher level in alkali-stressed wheat roots than in control and salt-stressed wheat roots, including 2 fatty acids, 3 amino acid derivatives, 1 dipeptide, 2 organic acids, 11 phenolic acids, 2 flavonoids, 1 lipid, 1 terpenoid, and 6 alkaloids. These alkali stress-induced accumulated carboxylic acids not only have roles in osmotic regulation but also directly or indirectly support root secretion during the response of wheat to alkali stress. The enhanced accumulation of carboxylic acids (e.g., amino acids, fatty acids, and organic acids) has also been observed in alkali-stressed rice [19], alfalfa [9], and sunflower [37]. RNAseq analysis showed that 16 key fatty acid synthesis genes and 122 phenolic acid synthesis genes (including rate-limiting enzyme genes PAL) have a higher expression level in wheat roots under alkali stress conditions than under control and salt stress conditions (Figure S4), indicating a strategy for the regulation of gene expression for the accumulation and secretion of fatty acids and phenolic acids during the response of wheat roots to alkali stress. Additionally, the expression level of 18 NPF genes and 25 peptide transporter genes was higher in alkali-stressed wheat roots than in control and salt-stressed wheat roots (Figure S1). The NPF family can transport multiple substrates, including chloride, potassium, carboxylate, plant hormones, peptides, nitrate, and metabolites containing a –COOH group [38]. The upregulated expression of the NPF genes may accelerate the secretion of metabolites containing the –COOH group and facilitate rhizosphere pH regulation in alkali-stressed wheat. Although we have identified some candidate genes that can mediate root secretion of wheat plants under alkali stress, some important questions remain, such as which genes mediate the co-expression of 19 NPF genes and 25 peptide transporter genes under alkali stress and what mechanism coordinates the production and secretion of AISMs. In wheat plants, alkali stress-induced secreted dipeptides and amino acids may be produced from protein degradation, while other secreted carboxylic acids may be generated from continuous biosynthesis. The upregulation of E3 ubiquitin-protein ligase genes, ubiquitin-conjugating enzyme genes, and protease genes facilitates the protein degradation that generates oligopeptides or amino acids (Figure S6), which provides materials for the secretion of dipeptides and amino acids by wheat roots under alkali stress. In wheat roots, the expression of 11 key ethylene synthesis genes and 38 ethylene-responsive transcription factor genes was particularly upregulated under alkali stress condition, suggesting that ethylene may mediate the response of wheat roots to alkali stress. It has been reported that ethylene plays a beneficial role in enhancing the salt tolerance of plants [39]. Ethylene may exert important effects in mediating the production and secretion of carboxylic acids and dipeptides during the response of wheat roots to alkali stress, which warrants further investigations.
糖解作用為代謝過程和根部分泌作用提供了還原力(ATP 和 NADH)及碳源。在小麥根部，五個 6-磷酸果糖激酶(糖解作用限速酶)基因在鹼脅迫下的表現量高於對照組和鹽脅迫條件(圖 S3)。增強的糖解作用將為脂肪酸、酚酸和有機酸的合成提供更多還原力和碳源，以支持這些物質在小麥響應鹼脅迫期間分泌至根際。我們也關注了在鹼脅迫小麥根部含量高於對照組和鹽脅迫小麥根部的代謝物，包括 2 種脂肪酸、3 種胺基酸衍生物、1 種二肽、2 種有機酸、11 種酚酸、2 種黃酮類、1 種脂質、1 種萜類和 6 種生物鹼。這些由鹼脅迫誘導累積的羧酸不僅在滲透調節中發揮作用，也在小麥響應鹼脅迫期間直接或間接地支持根部分泌作用。 在鹼脅迫下的水稻[19]、苜蓿[9]和向日葵[37]中也觀察到羧酸（如胺基酸、脂肪酸和有機酸）的累積增加現象。RNAseq 分析顯示，與對照組和鹽脅迫條件相比，小麥根部在鹼脅迫條件下有 16 個關鍵脂肪酸合成基因和 122 個酚酸合成基因（包括限速酶基因 PAL）表現量更高（圖 S4），這表明小麥根部在應對鹼脅迫時，存在調控脂肪酸和酚酸累積與分泌的基因表現策略。此外，鹼脅迫下小麥根部有 18 個 NPF 基因和 25 個肽轉運蛋白基因的表現量高於對照組和鹽脅迫組（圖 S1）。NPF 家族能轉運多種底物，包括氯離子、鉀離子、羧酸鹽、植物激素、肽類、硝酸鹽以及含有–COOH 基團的代謝物[38]。NPF 基因的上調表現可能加速含有–COOH 基團代謝物的分泌，有助於鹼脅迫下小麥根際 pH 值的調節。 儘管我們已鑑定出一些能夠調控小麥植株在鹼脅迫下根部分泌的候選基因，但仍有若干關鍵問題尚待釐清，例如哪些基因介導了 19 個 NPF 基因與 25 個肽轉運蛋白基因在鹼脅迫下的共表達現象，以及何種機制協調了 AISMs 的生成與分泌過程。在小麥植株中，鹼脅迫誘導分泌的二肽與胺基酸可能源自蛋白質降解途徑，而其他分泌的有機酸類則可能透過持續的生物合成途徑產生。E3 泛素蛋白連接酶基因、泛素接合酶基因及蛋白酶基因的上調表現，促進了生成寡肽或胺基酸的蛋白質降解過程（圖 S6），這為小麥根部在鹼脅迫下分泌二肽與胺基酸提供了物質基礎。研究發現，小麥根部在鹼脅迫條件下特別顯著上調表現了 11 個關鍵乙烯合成基因與 38 個乙烯響應轉錄因子基因，暗示乙烯訊號可能介導了小麥根部對鹼脅迫的應答機制。 已有研究指出，乙烯在增強植物耐鹽性方面發揮有益作用[39]。乙烯可能透過調控羧酸與二肽的生成與分泌，在小麥根系應對鹼脅迫反應中產生重要影響，此機制值得進一步研究探討。

4. Materials and Methods
4. 材料與方法

4.1. Stress Treatment and Root Exudate Collection
4.1. 逆境處理與根系分泌物收集

Xiaobingmai33, a spring wheat variety widely cultivated in Northeast China, was selected as the test organism. The wheat seeds were provided by Prof. Jinsong Pang from Northeast Normal University, China. The seeds were sown in plastic pots containing sand. All pots (15 seedlings per pot; pot size height 19 cm and diameter 18.5 cm) were watered with half-strength Hoagland nutrient solution for 30 days in a greenhouse (23–25 °C day and 17–20 °C night, 16 h light). The experiment was conducted from mid-April to mid-May in Changchun, China. Based on the pH and salinity levels of moderate soda salt-alkaline in Northeast China, NaHCO₃ and Na₂CO₃ were added at a 9:1 molar ratio (80 mM total salt concentration, 88 mM Na⁺ concentration, and pH 8.8) to mimic alkali stress conditions in the moderate soda salt–alkaline soil. To explore the specific effects of high-pH, NaCl and Na₂SO₄ were added at a 9:1 molar ratio (80 mM total salt concentration, 88 mM Na⁺ concentration, and pH 6.7) for the salt stress treatment. The control was cultured with a half-strength Hoagland nutrient solution (pH 6.6). The final pH values of the salt stress and alkali stress treatment solutions were determined after adding the nutrient solution. Wheat plants can finish their life cycle under such stress conditions. The pots with uniform wheat seedlings were treated with salt or alkali treatment solution containing nutrient components for three days, and then root exudates were collected and stored at −80 °C according to a method by Li et al. [33]. After root exudate collection, the root samples were collected and freeze-dried, and RNA samples were collected and stored at −80 °C. Ten plants were pooled as a biological replicate, with three biological replicates for metabolome analysis and RNA sequencing.
選用中國東北地區廣泛種植的春小麥品種「小冰麥 33 號」作為試驗材料。小麥種子由中國東北師範大學龐金聲教授提供。種子播種於裝有沙質基質的塑膠盆中。所有盆器（每盆 15 株幼苗；盆器尺寸高 19 公分、直徑 18.5 公分）在溫室條件下（日溫 23-25°C、夜溫 17-20°C，每日光照 16 小時）以半濃度霍格蘭營養液澆灌 30 天。實驗於中國長春進行，時間為四月中旬至五月中旬。根據中國東北地區中度蘇打鹽鹼土的 pH 值與鹽分濃度，以 9:1 莫耳比例添加 NaHCO₃與 Na₂CO₃（總鹽濃度 80 mM，Na⁺濃度 88 mM，pH 值 8.8）模擬中度蘇打鹽鹼土的鹼性逆境條件。為探究高 pH 值的特定效應，另以 9:1 莫耳比例添加 NaCl 與 Na₂SO₄（總鹽濃度 80 mM，Na⁺濃度 88 mM，pH 值 6.7）作為鹽分逆境處理組。 對照組使用半濃度霍格蘭營養液(pH 6.6)進行培養。鹽脅迫與鹼脅迫處理液的最終 pH 值於添加營養液後測定。小麥植株在此脅迫條件下能完成其生命週期。將生長均勻的小麥幼苗盆栽分別以含營養成分的鹽處理液或鹼處理液處理三天後，依據 Li 等人[33]的方法收集根系分泌物並儲存於-80°C。根系分泌物採集完成後，收取根部樣本進行冷凍乾燥，同時採集 RNA 樣本儲存於-80°C。每 10 株植株混合為一個生物學重複，代謝體學分析與 RNA 定序各設置三個生物學重複。

4.2. Metabolome Analysis  4.2. 代謝體學分析

Metabolites in root exudates and root tissues were qualified and quantified using a widely targeted metabolomics approach based on a local MS-MS data library constructed with authentic standards [40]. The secretion rate of each metabolite was expressed as the relative amount (peak area) of g⁻¹ root DW. Metabolites in root exudates and root tissues were measured according to Li et al. [33]. Briefly, freeze-dried root samples and freeze-dried root exudates were treated with 70% methanol, and then the extracts were loaded onto an LC–MS/MS system (QTRAP, AB SCIEX). A mixed sample of all extracts in equal volumes was loaded onto an LC–MS/MS system (QTRAP, AB SCIEX) to construct an MS2 spectral tag library. Retention time, m/z ratio, and fragmentation information were applied to identify each metabolite through an in-house database (MWDB, https://www.metware.cn accessed on 11 December 2021). All the metabolites identified were quantified using the MRM method [40]. We defined differentially accumulated metabolite (DAM) or differentially secreted metabolite (DSM) as VIP > 1, p value (t test) < 0.05, and |Log2(Fold change)| > 1.
採用基於真實標準品建立的本地 MS-MS 數據庫[40]，透過廣泛靶向代謝組學方法對根系分泌物及根組織中的代謝物進行定性與定量分析。各代謝物分泌速率以每克根乾重(g ⁻¹ root DW)的相對含量(峰面積)表示。根系分泌物與根組織代謝物測定參照 Li 等人[33]方法：將冷凍乾燥的根樣品與冷凍乾燥的根系分泌物經 70%甲醇處理後，萃取液導入 LC-MS/MS 系統(QTRAP, AB SCIEX)；另取等體積混合萃取液建立 MS2 光譜標籤庫。透過保留時間、質荷比(m/z)及碎片資訊，利用內部數據庫(MWDB, https://www.metware.cn 2021 年 12 月 11 日存取)鑑定代謝物，並採用多反應監測模式(MRM)進行定量[40]。差異累積代謝物(DAM)與差異分泌代謝物(DSM)的篩選標準為：變數重要性投影(VIP)>1、p 值(t 檢定)<0.05 且|Log2(倍數變化)|>1。

4.3. RNAseq and qRT-PCR  4.3. 轉錄組測序(RNAseq)與即時定量 PCR(qRT-PCR)

Conventional methods were applied to conduct RNAseq experiments and data analyses [2]. Total RNA samples were used as input material for library construction. The prepared libraries were sequenced on an Illumina platform. Wheat reference genome and gene model annotation files were downloaded from the Ensembl Plants website (http://plants.ensembl.org/Triticum_aestivum/Info/Index accessed on 20 December 2021). The paired-end clean reads were mapped to the reference genome using Hisat2 v2.0.5. Differentially expressed genes (DEGs) were identified using the DESeq2 R package 1.20.0 (adjusted p value ≤ 0.05 and |log2fold change| ≥ 1) [41]. We applied the TBtools program to conduct GO and KEGG enrichments for DEGs [42]. The reliability of the RNAseq analysis was validated using qRT-PCR. RLI, Actin 2, Actin 7, and β-tubulin were selected as internal control genes. The expression level of the genes was calculated using the ΔΔCt method [43].
本研究採用常規方法進行 RNAseq 實驗與數據分析[2]。以總 RNA 樣本作為文庫構建的輸入材料，製備的文庫於 Illumina 平台進行測序。小麥參考基因組及基因模型註釋文件下載自 Ensembl Plants 網站(http://plants.ensembl.org/Triticum_aestivum/Info/Index，存取日期 2021 年 12 月 20 日)。使用 Hisat2 v2.0.5 將雙端乾淨讀段比對至參考基因組，並透過 DESeq2 R 套件 1.20.0 鑑定差異表現基因(DEGs)(調整後 p 值≤0.05 且|log2 倍數變化|≥1)[41]。運用 TBtools 程式進行 DEGs 的 GO 與 KEGG 富集分析[42]。以 qRT-PCR 驗證 RNAseq 分析可靠性，選用 RLI、Actin 2、Actin 7 及β-tubulin 作為內參基因，採用ΔΔCt 法計算基因表現量[43]。

5. Conclusions  5. 結論

The secretion of multiple types of metabolites with a –COOH group is an important pH regulation strategy for alkali-stressed wheat plants. Enhanced glycolysis, fatty acid synthesis, and phenolic acid synthesis will provide more energy and substrates for root secretion during the response of wheat to alkali stress. In wheat plants, alkali stress-induced secreted dipeptides and amino acids may be produced from protein degradation, while other secreted carboxylic acids may be generated from continuous biosynthesis. Some NPF genes and peptide transporter genes may play important roles in the pH regulation of alkali-stressed wheat plants.
分泌多種帶有-COOH 基團的代謝物是小麥植株應對鹼脅迫的重要 pH 調節策略。在響應鹼脅迫過程中，增強糖酵解、脂肪酸合成和酚酸合成將為根部分泌提供更多能量和基質。在小麥植株中，鹼脅迫誘導分泌的二肽和胺基酸可能來自蛋白質降解，而其他分泌的有機酸則可能來自持續的生物合成。某些 NPF 基因和肽轉運蛋白基因可能在鹼脅迫小麥植株的 pH 調節中發揮重要作用。