Screening of Upstream Transcription Factors of MeGLYI-13 Gene Related to Postharvest Physiological Deterioration of Cassava

doi:10.13560/j.cnki.biotech.bull.1985.2025-1343

Abstract

Abstract:

Objective Cassava (Manihot esculenta Crantz) is one of the most important economic and food crops in the world. The problem of postharvest physiological deterioration (PPD) causes a serious impact on the storage period and economic benefits. As a member of the glyoxalaseⅠ (GLYⅠ) family, the expressionof MeGLYI-13 was positively correlated with the PPD tolerance of cassava. In-depth analysis of the molecular mechanism of upstream regulation of MeGLYI-13 involved in cassava PPD tolerance may provide a new idea for exploring the molecular mechanism of cassava postharvest physiological deterioration process, and PPD-tolerant cassava germplasm innovation. Method The promoter region sequence of MeGLYI-13 was cloned and analyzed. The proMeGLYI-13-pAbAi bait vector was constructed and transformed into yeast cells to obtain the bait strain. The expression pattern analysis and interaction verification of candidate transcription factors were carried out by yeast one-hybrid screening library experiment using cassava cDNA library. Result The 2 000 bp upstream promoter sequence of MeGLYI-13 was successfully cloned. The sequence contained cis-acting elements responding to low temperature, drought, auxin, gibberellin, light, defense and stress. Through yeast one-hybrid screening, four candidate transcription factors were screened from cassava cDNA library, which were ethylene-responsive transcription factor ERF1 and AT-hook motif nuclear localization proteins AHL17, AHL29 and AHL31. The rotary verification test and dual luciferase test further confirmed that MeAHL17 played a negative regulatory role in the promoter activity of MeGLYI-13. Gene expression analysis showed that the expression trend of MeAHL17 was negatively correlated with PPD tolerance of cassava. Conclusion The combination of MeAHL17 and MeGLYI-13 promoter regulate gene expression, which may play a role in the physiological deterioration of cassava after harvest.

Key words: cassava, postharvest physiological deterioration, glyoxalase, transcription factors, promoter, AT-hook motif nuclear localization protein

SHEN Chen, CHE Yan-nian, DING Zhong-ping, WANG Xiang-wen, GE Yu-jian, FU Dong-qing, ZHOU Ya-xing-qiao, LI Rui-mei. Screening of Upstream Transcription Factors of MeGLYI-13 Gene Related to Postharvest Physiological Deterioration of Cassava[J]. Biotechnology Bulletin, 2026, 42(6): 98-106.

Figures/Tables 9

Table 1 Related information of the truncated fragment of MeGLYI-13 promoter

名称

Name

在启动子序列中

起始位置

The start positions in the promoter sequence （bp）

在启动子序列中

终止位置

The end positions in the promoter sequence （bp）

片段长度

Fragment length （bp）

顺式作用元件

Cis-acting elements

ARE， TCCC-motif， MBS， LTR， P-box， Box 4， CAT-box

LTR， MRE， GA-motif， CAT-box， TGA-element

TCT-motif， GT1-motif， TC-rich repeats， Sp1， GATA-motif

Fig. 1 Partial cis-acting elements of MeGLYI-13 gene promoter

Fig. 2 Screening of AbA inhibitory concentration of bait strainp53-pAbAi: Positive control. proMeGLYI-13-pAbAi: Bait strain

Fig. 3 The second screening of AbA concentration of bait yeast strain

Fig. 4 Candidate positive plaque screening and plaque PCR verificationM: 2 000 marker. A-B: Seg1 screening of transcription factors. C-F: Seg2 screening of transcription factors

Table 2 Information of candidate transcription factors screened by proMeGLY13 promoter

序号 Number	登录号 Gene ID	名称 Name	功能注释 Functional annotation
1	Manes.02G145600	AHL17	AT-hook motif nuclear-localized protein 17
2	Manes.09G149500	AHL29	AT-hook motif nuclear-localized protein 29
3	Manes.15G039700	ERF1	Ethylene-responsive transcription factor 1
4	Manes.10G027000	AHL31	AT-hook motif nuclear-localized protein 31

Fig. 5 Rotational verification of MeGLYI-13 promoter and candidate transcription factors

Fig. 6 Transcriptome expression analysis and verification of MeGLYI-13, MeAHL17, MeAHL29 and MeAHL31 in theroots after harvest

Fig. 7 Dual-luciferase assay to verify the relationship between MeAHL17and the promoter of MeGLYI-13

References 42

[1]	Falade KO, Akingbala JO. Utilization of cassava for food [J]. Food Rev Int, 2010, 27(1): 51-83.
[2]	An D, Yang J, Zhang P. Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress [J]. BMC Genom, 2012, 13: 64.
[3]	Iyer S, Mattinson DS, Fellman JK. Study of the early events leading to cassava root postharvest deterioration [J]. Trop Plant Biol, 2010, 3(3): 151-165.
[4]	Ephraim N, Patrick RR, Ssetumba M, et al. Biochemical and secondary metabolites changes under moisture and temperature stress in cassava (Manihot esculenta Crantz) [J]. Afr J Biotechnol, 2014, 13(31): 3173-3186.
[5]	Raza H, Khan MR, Zafar SA, et al. Aldehyde dehydrogenase 3I1 gene is recruited in conferring multiple abiotic stress tolerance in plants [J]. Plant Biol J, 2022, 24(1): 85-94.
[6]	Sunkar R, Bartels D, Kirch HH. Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance [J]. Plant J, 2003, 35(4): 452-464.
[7]	Hoque TS, Hossain MA, Mostofa MG, et al. Methylglyoxal: an emerging signaling molecule in plant abiotic stress responses and tolerance [J]. Front Plant Sci, 2016, 7: 1341.
[8]	Sankaranarayanan S, Jamshed M, Samuel MA. Degradation of glyoxalase I in Brassica napus stigma leads to self-incompatibility response [J]. Nat Plants, 2015, 1(12): 15185.
[9]	Ye XY, Qiu XM, Sun YY, et al. Interplay between hydrogen sulfide and methylglyoxal initiates thermotolerance in maize seedlings by modulating reactive oxidative species and osmolyte metabolism [J]. Protoplasma, 2020, 257(5): 1415-1432.
[10]	Liu SJ, Liu WH, Lai JY, et al. OsGLYI3, a glyoxalase gene expressed in rice seed, contributes to seed longevity and salt stress tolerance [J]. Plant Physiol Biochem, 2022, 183: 85-95.
[11]	Gambhir P, Raghuvanshi U, Parida AP, et al. Elevated methylglyoxal levels inhibit tomato fruit ripening by preventing ethylene biosynthesis [J]. Plant Physiol, 2023, 192(3): 2161-2184.
[12]	Mostofa MG, Ghosh A, Li ZG, et al. Methylglyoxal-a signaling molecule in plant abiotic stress responses [J]. Free Radic Biol Med, 2018, 122: 96-109.
[13]	Engqvist M, Drincovich MF, Flügge UI, et al. Two D-2-hydroxy-acid dehydrogenases in Arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and β-oxidation pathways [J]. J Biol Chem, 2009, 284(37): 25026-25037.
[14]	Ali S, Rizwan M, Arif MS, et al. Approaches in enhancing thermotolerance in plants: an updated review [J]. J Plant Growth Regul, 2020, 39(1): 456-480.
[15]	Wienstroer J, Engqvist MKM, Kunz HH, et al. D-Lactate dehydrogenase as a marker gene allows positive selection of transgenic plants [J]. FEBS Lett, 2012, 586(1): 36-40.
[16]	Jin SH, Li XQ, Wang GG, et al. Brassinosteroids alleviate high-temperature injury in Ficus concinna seedlings via maintaining higher antioxidant defence and glyoxalase systems [J]. Aob PLANTS, 2015, 7: plv009.
[17]	Nahar K, Hasanuzzaman M, Alam MM, et al. Insights into spermine-induced combined high temperature and drought tolerance in mung bean: osmoregulation and roles of antioxidant and glyoxalase system [J]. Protoplasma, 2017, 254(1): 445-460.
[18]	Zhang YM, Yang CY, Liu SX, et al. Alleviation of salt toxicity by melatonin supplementation in eggplant involves the up-regulation of ascorbate-glutathione and glyoxalase system [J]. Sci Hortic, 2024, 326: 112720.
[19]	Li TM, Cheng X, Wang XW, et al. Glyoxalase I-4 functions downstream of NAC72 to modulate downy mildew resistance in grapevine [J]. Plant J, 2021, 108(2): 394-410.
[20]	Duan M, Zhang RX, Zhu FG, et al. A lipid-anchored NAC transcription factor is translocated into the nucleus and activates Glyoxalase I expression during drought stress [J]. Plant Cell, 2017, 29(7): 1748-1772.
[21]	Hasanuzzaman M, Alam MM, Nahar K, et al. Silicon-induced antioxidant defense and methylglyoxal detoxification works coordinately in alleviating nickel toxicity in Oryza sativa L [J]. Ecotoxicology, 2019, 28(3): 261-276.
[22]	Li RM, Tang FL, Che YN, et al. MeGLYI-13, a glyoxalase I gene in cassava, enhances the tolerance of yeast and Arabidopsis to Zinc and copper stresses [J]. Plants, 2023, 12(19): 3375.
[23]	Tang FL, Li RM, Zhou YJ, et al. Genome-wide identification of cassava glyoxalase I genes and the potential function of MeGLYⅠ-13 in iron toxicity tolerance [J]. Int J Mol Sci, 2022, 23(9): 5212.
[24]	唐芬莲. 木薯乙二醛酶基因MeGLYI-13的克隆及功能分析 [D]. 海口: 海南大学, 2022.
	Tang FL. Cloning and functional analysis of cassava glyoxalase gene MeGLYI-13 [D]. Haikou: Hainan University, 2022.
[25]	胡文斌. 木薯可溶性淀粉合成酶基因IV启动子的功能鉴定及其调控因子的筛选 [D]. 海口: 海南大学, 2013.
	Hu WB. Functional identification of cassava soluble starch synthase gene IV promoter and screening of its regulatory factors [D]. Haikou: Hainan University, 2013.
[26]	Li RM, Yuan S, Zhou YJ, et al. Comparative transcriptome profiling of cassava tuberous roots in response to postharvest physiological deterioration [J]. Int J Mol Sci, 2022, 24(1): 246.
[27]	Smits MM, Johnson MA. Methylglyoxal: Enzyme distributions relative to its presence in douglas-fir needles and absence in douglas-fir needle callus [J]. Arch Biochem Biophys, 1981, 208(2): 431-439.
[28]	Kaur C, Ghosh A, Pareek A, et al. Glyoxalases and stress tolerance in plants [J]. Biochem Soc Trans, 2014, 42(2): 485-490.
[29]	Sankaranarayanan S, Jamshed M, Kumar A, et al. Glyoxalase goes green: the expanding roles of glyoxalase in plants [J]. Int J Mol Sci, 2017, 18(4): 898.
[30]	Zhao JF, Favero DS, Qiu JW, et al. Insights into the evolution and diversification of the AT-hook Motif Nuclear Localized gene family in land plants [J]. BMC Plant Biol, 2014, 14: 266.
[31]	Wang M, Chen BW, Zhou W, et al. Genome-wide identification and expression analysis of the AT-hook Motif Nuclear Localized gene family in soybean [J]. BMC Genom, 2021, 22: 361.
[32]	Grasser M, Christensen JM, Peterhänsel C, et al. Basic and acidic regions flanking the HMG-box domain of maize HMGB1 and HMGB5 modulate the stimulatory effect on the DNA binding of transcription factor Dof2 [J]. Biochemistry, 2007, 46(21): 6375-6382.
[33]	Xiao CW, Chen FL, Yu XH, et al. Over-expression of an AT-hook gene, AHL22 delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana [J]. Plant Mol Biol, 2009, 71(1/2): 39-50.
[34]	Ambadas DA, Singh A, Jha RK, et al. Genome-wide dissection of AT-hook motif nuclear-localized gene family and their expression profiling for drought and salt stress in rice (Oryza sativa) [J]. Front Plant Sci, 2023, 14: 1283555.
[35]	Dong JH, Xu LB, Zeng HY, et al. Genome-wide identification and analyses of the AHL gene family in banana (Musa acuminata) and the functional analysis of MaAHL43 in fruit ripening [J]. Sci Hortic, 2025, 341: 113948.
[36]	Favero DS, Kawamura A, Shibata M, et al. AT-hook transcription factors restrict petiole growth by antagonizing PIFs [J]. Curr Biol, 2020, 30(8): 1454-1466.
[37]	Zhou LG, Liu ZC, Liu YH, et al. A novel gene OsAHL1 improves both drought avoidance and drought tolerance in rice [J]. Sci Rep, 2016, 6: 30264.
[38]	Yadeta KA, Hanemian M, Smit P, et al. The Arabidopsis thaliana DNA-binding protein AHL19 mediates Verticillium wilt resistance [J]. Mol Plant Microbe Interact, 2011, 24(12): 1582-1591.
[39]	Wang LY, Li TT, Liu N, et al. Identification of tomato AHL gene families and functional analysis their roles in fruit development and abiotic stress response [J]. Plant Physiol Biochem, 2023, 202: 107931.
[40]	Shi XW, Yang T, Ren MF, et al. AT-hook motif nuclear localized transcription factors function redundantly in promoting root growth through modulation of redox homeostasis [J]. Plant J, 2024, 120(1): 199-217.
[41]	张亚文, 王晓彤, 张兴龙, 等. 木薯MeAHL17基因的克隆及表达分析 [J]. 热带作物学报, 2024, 45(7): 1303-1313.
	Zhang YW, Wang XT, Zhang XL, et al. Cloning and expression analysis of MeAHL17 gene in cassava [J]. Chin J Trop Crops, 2024, 45(7): 1303-1313.
[42]	Bai RY, Zhou MM, Zheng Y, et al. Transcription factor AHL17 fine-tunes reactive oxygen species accumulation and cassava disease resistance [J]. Plant Physiol, 2024, 195(2): 1148-1151.