Data Sheet 1_Nitric oxide-mediated modulation of reproductive resilience under cold stress in chickpea.docx

Chickpeas are particularly sensitive to cold stress during the reproductive phase, which can significantly impair pod set and yield. This study examined the role of sodium nitroprusside (SNP), a nitric oxide (NO) donor, in mitigating cold-induced reproductive damage in cold-tolerant (CT) and cold-sensitive (CS) chickpea genotypes. After 100 days of outdoor growth, plants were subjected to cold stress (15/8°C day/night; 12 h photoperiod) for 21 days in walk-in growth chambers during the reproductive stage of development. Control plants were maintained at 25/15°C day/night temperature. SNP treatment (1 mM) was applied exogenously each time, first two days prior to stress onset and then at seven-day intervals (three applications total). Cold stress significantly lowered endogenous NO levels in leaves, anthers, and ovules, particularly in CS genotypes, thereby leading to reduced pollen viability and germination. SNP treatment restored NO and improved reproductive performance, with stronger responses in the CS than the CT genotype. For instance, pollen germination increased by 57.9% in CS versus 17.6% in CT, and pollen viability increased by 28.0% and 13.1%, respectively. Enhanced anther function resulted in a 157.2% increase in pod set and 62.0% higher seed yield in CS. SNP also improved physiological traits, including a 43.9% increase in cellular viability, 18.6% in stomatal conductance, and 41.9% in chlorophyll content in CS genotypes. Cryoprotectants (proline, trehalose, and sucrose) accumulated in anthers, reinforcing cold resilience, while oxidative stress was simultaneously alleviated through reduced malondialdehyde, hydrogen peroxide, and electrolyte leakage, together with the upregulation of both enzymatic (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx), and glutathione reductase (GR)) and non-enzymatic (ascorbic acid (ASC) and reduced glutathione (GSH)) components. Notably, CS genotypes showed more pronounced improvements from SNP application than CT genotypes, particularly in terms of reproductive success and yield-related traits. These findings highlight the potential of NO donors, such as SNP, to enhance cold tolerance in chickpeas, with promising implications for safeguarding productivity under low-temperature stress, especially in sensitive cultivars.

鹰嘴豆在生殖期对冷胁迫尤为敏感，冷胁迫会显著损害其结荚率与籽粒产量。本研究探讨了硝普钠（sodium nitroprusside, SNP）——一种一氧化氮（nitric oxide, NO）供体——在缓解耐冷（cold-tolerant, CT）与冷敏感（cold-sensitive, CS）鹰嘴豆基因型冷诱导生殖损伤中的作用。试验于植株生殖生长阶段在人工气候培养箱中开展：先将露地生长100天的植株置于15/8℃（昼/夜）、12小时光周期的环境下进行为期21天的冷胁迫处理；对照组植株则维持在25/15℃（昼/夜）的温度条件。每次外源施加1 mM浓度的SNP溶液，分别于冷胁迫开始前2天、胁迫开始后每7天施加一次，共施药3次。冷胁迫显著降低了叶片、花药与胚珠中的内源NO水平，在冷敏感基因型中这一现象尤为突出，进而导致花粉活力与萌发率下降。SNP处理可恢复内源NO水平并改善生殖性能，且冷敏感基因型的响应效果优于耐冷基因型。例如，冷敏感基因型的花粉萌发率提升57.9%，耐冷基因型仅提升17.6%；花粉活力分别提升28.0%与13.1%。花药功能的增强使冷敏感基因型的结荚率提升157.2%，籽粒产量提升62.0%。SNP还改善了多项生理性状：冷敏感基因型的细胞活力提升43.9%，气孔导度提升18.6%，叶绿素含量提升41.9%。花药中积累了抗寒保护剂（脯氨酸、海藻糖与蔗糖），增强了植株的耐寒性；同时通过降低丙二醛、过氧化氢含量与电解质渗漏率，并上调酶促抗氧化系统（超氧化物歧化酶（superoxide dismutase, SOD）、过氧化氢酶（catalase, CAT）、抗坏血酸过氧化物酶（ascorbate peroxidase, APx）与谷胱甘肽还原酶（glutathione reductase, GR））及非酶促抗氧化系统（抗坏血酸（ascorbic acid, ASC）与还原型谷胱甘肽（reduced glutathione, GSH））的表达，有效缓解了氧化胁迫。值得注意的是，冷敏感基因型经SNP处理后的改善效果较耐冷基因型更为显著，尤其是在生殖成功率与产量相关性状方面。本研究结果表明，硝普钠这类一氧化氮供体具备提升鹰嘴豆耐寒性的潜力，可为低温胁迫下的作物生产力保障提供可行途径，尤其对冷敏感品种具有重要应用价值。