Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

拟南芥CBF1与植物对低温和干旱的抗性

对冷驯化过程中基因表达差异的认识,使抗冻基因(COR)的克隆及其功能的分析成为研究冷驯化过程的主要目标。在拟南芥和其他抗冻植物中,分离了许多COR基因,这些基因对植物抗冻起着非常重要的作用。在拟南芥COR调控的研究中,发现了CBF转录因子的基因家族,其中CBF1能调控一组COR基因的表达。近年来,在冷敏植物如番茄和玉米中也发现了CBF类似基因,拟南芥CBF1基因在转基因番茄中的过量表达提高了植株的抗寒和抗旱性。这一研究结果展示了拟南芥CBF1类似基因的应用可能为冷敏植物抗寒和抗旱性的品种改良提供一条新的途径。     

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.     

组成型表达的ClpB基因提高番茄植株的耐冷性

用农杆菌介导法将CaMV35S启动子驱动的ClpB cDNA导入番茄,并比较了转基因和未转基因番茄的抗冷能力。当受冷胁迫后,转基因番茄比未转基因番茄表现出较轻的冷胁迫症状,并维持较高的PSII水平。     

外源水杨酸对低温下杏花抗氧化酶和CBF转录因子表达的影响

为探讨水杨酸（SA）对杏花抗寒性的影响机制,以早熟品种‘骆驼黄’杏的显蕾期花枝为试材,分析–2℃的低温下适宜浓度SA及其抑制剂ABT和PAC对杏花MDA、抗氧化酶和CBF转录因子的影响。结果表明,–2℃低温条件下,对照和2个SA抑制剂处理的杏花细胞膜系统均受到严重伤害,CAT、POD和SOD等抗氧化酶活性降低,MDA含量明显升高。而SA预处理的杏花在低温胁迫期间抗氧化酶活性增强,MDA含量比对照和抑制剂处理的有明显降低且相对稳定。通过荧光定量检测CBF转录因子的表达水平,表明SA能诱导杏花CBF基因的表达,尤其在低温处理3 h时,SA预处理的杏花中CBF的表达量明显高于对照和SA抑制剂处理。由此认为,适宜浓度的外源SA可能是通过调控低温下杏花中CBF转录因子的表达、增强细胞的抗氧化酶活性,减轻低温造成的膜脂过氧化伤害,从而在一定程度上增强了杏花的抗寒性。     

New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome‐wide association analysis

In order to understand cold adaptability and explore additional genetic resources for the cold tolerance improvement of rice, we investigated the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome‐wide association studies; a total of 132 loci were identified. Among them, 12 loci were common for both chilling and cold shock tolerance, suggesting that rice has a distinct and overlapping genetic response and adaptation to the two stresses. Haplotype analysis of a known gene OsMYB2, which is involved in cold tolerance, revealed indica–japonica differentiation and latitude tendency for the haplotypes of this gene. By checking the subpopulation and geographical distribution of accessions with tolerance or sensitivity under these two stress conditions, we found that the chilling tolerance group, which mainly consisted of japonica accessions, has a wider latitudinal distribution than the chilling sensitivity group. We conclude that the genetic basis of natural chilling stress tolerance in rice is distinct from that of cold shock stress frequently used for low‐temperature treatment in the laboratory and the cold adaptability of rice is associated with the subpopulation and latitudinal distribution.     

Putrescine enhances chilling tolerance of tomato (Lycopersicon esculentum Mill.) through modulating antioxidant systems

Seedlings of two tomato (Lycopersicon esculentum Mill.) cultivars, cv. Mawa (chilling-resistant) and cv. Moneymaker (chilling-sensitive) were used to investigate the effects of exogenous putrescine (Put) on chilling tolerance as well as on changes of physiological features and the fluctuation of free and conjugated endogenous polyamines (PAs) contents in the leaves under chilling stress. During chilling stress, accumulation of hydrogen peroxide (H₂O₂) was obviously detected in the leaves of both cultivars, but it was fewer in cv. Mawa. Meanwhile, d-arginine (d-Arg), a Put biosynthesis inhibitor caused more H₂O₂ accumulation in both cultivars, especially in cv. Moneymaker. By adding back Put to leaves, accumulation of H₂O₂ obviously reduced in two cultivars. Put was also involved in the increase of Fv/Fm and the decrease of malondialdehyde (MDA) in two cultivars under chilling stress. Despite the two cultivars displaying differential behavior towards enzymic antioxidants, enzymes and components of the ascorbate–glutathione (AsA–GSH) cycle in responses to chilling stress, d-Arg treatment diminished the enzyme activities and antioxidant contents induced by chilling stress and its reversion was performed by adding Put in both cultivars. During chilling stress, free and conjugated endogenous PA contents increased in two cultivars. d-Arg treatment inhibited the increases, and exogenously applied Put enhanced the increases in two cultivars. These results suggested that Put played important roles in the tolerance of tomato against chilling stress, which was most likely achieved by modulating antioxidant system as well as increasing free and conjugated PAs.     

CbCBF from Capsella bursa-pastoris enhances cold tolerance and restrains growth in Nicotiana tabacum by antagonizing with gibberellin and affecting cell cycle signaling

Plant cells respond to cold stress via a regulatory mechanism leading to enhanced cold acclimation accompanied by growth retardation. The C-repeat binding factor (CBF) signaling pathway is essential for cold response of flowering plants. Our previously study documented a novel CBF-like gene from the cold-tolerant Capsella bursa-pastoris named CbCBF, which was responsive to chilling temperatures. Here, we show that CbCBF expression is obviously responsive to chilling, freezing, abscisic acid, gibberellic acid (GA), indoleacetic acid or methyl jasmonate treatments and that the CbCBF:GFP fusion protein was localized to the nucleus. In addition, CbCBF overexpression conferred to the cold-sensitive tobacco plants enhanced tolerance to chilling and freezing, as well as dwarfism and delayed flowering. The leaf cells of CbCBF overexpression tobacco lines attained smaller sizes and underwent delayed cell division with reduced expression of cyclin D genes. The dwarfism of CbCBF transformants can be partially restored by GA application. Consistently, CbCBF overexpression reduced the bioactive gibberellin contents and disturbed the expression of gibberellin metabolic genes in tobacco. Meanwhile, cold induced CbCBF expression and cold tolerance in C. bursa-pastoris are reduced by GA. We conclude that CbCBF confers cold resistance and growth inhibition to tobacco cells by interacting with gibberellin and cell cycle pathways, likely through activation of downstream target genes.