Transcriptome-wide Identification of the GRAS Gene Family in Mulberry and Analysis of MnGRAS22 in Response to Salt Stress

doi:10.13560/j.cnki.biotech.bull.1985.2025-1191

Abstract

Abstract:

Objective This study aimed to identify members of the MnGRAS gene family in mulberry (Morus alba), clarify their expression patterns and functional characteristics in response to salt stress, and provide theoretical support for the genetic improvement of salt tolerance in mulberry. Method MnGRAS genes were screened from mulberry transcriptome data under salt stress. Bioinformatic analyses were conducted to characterize their physicochemical properties, phylogenetic classification, gene structural features, and promoter cis-acting elements. Gene expression patterns were verified by integrating transcriptome data with RT-qPCR. The overexpression and RNA interference (RNAi)-mediated suppression systems of MnGRAS22 were constructed, and its salt-tolerance function was elucidated by determining physiological and biochemical indices. Result A total of 40 MnGRAS genes were successfully identified and classified into 10 subfamilies, with all members containing the typical GRAS domain. Promoter regions were enriched with cis-acting elements associated with growth, development, and abiotic stress responses. Under salt stress, 92.5% genes showed detectable expression signals, while only a few genes (MnGRAS11, MnGRAS27, MnGRAS34) had no expression at any stress time point. Among the 9 candidate genes, 66.7% were continuously upregulated in the leaves, whereas most showed dynamic expression patterns in the roots and stems. Specifically, the expressions of MnGRAS22 in the leaves and roots were 88-fold and 36-fold higher than those in the control, respectively. The overexpression of MnGRAS22 activated the expressions of oxidase genes (SOD, POD), significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in plants, promoted proline accumulation, and reduced the contents of malondialdehyde (MDA), superoxide anion radical (O₂^•-), and hydrogen peroxide (H₂O₂). Conclusion Forty MnGRAS genes were identified in mulberry, among which MnGRAS22 was highly expressed in the leaves and roots under salt stress. The overexpression of MnGRAS22 can significantly enhance plant salt tolerance by improving antioxidant capacity, regulating osmotic balance, and alleviating membrane damage.

Key words: mulberry, GRAS gene family, MnGRAS22, transcriptome, salt stress, gene identification, antioxidant response, salt tolerance

DONG Ya-ru, XIU Yu, ZHAO Dong-xiao, ZHU Hong, ZHU Lin, LIU Hui-fen. Transcriptome-wide Identification of the GRAS Gene Family in Mulberry and Analysis of MnGRAS22 in Response to Salt Stress[J]. Biotechnology Bulletin, 2026, 42(7): 193-203.

Figures/Tables 8

Fig. 1 Phylogenetic analysis of GRAS family members from mulberry and ArabidopsisThe red stars indicates the GRAS of mulberry and black triangle indicates Arabidopsis, respectively

Fig. 2 Conserved motif (A), domains (B), and gene structure (C) analysis of MnGRAS gene family in mulberry

Fig. 3 Distribution of cis-acting elements of GRAS gene promoter in mulberry

Fig. 4 Expression patterns of GRAS in mulberry in response to salt stressHeatmap showing expressions of GRAS genes in mulberry leaves under 200 mmol/L NaCl treatment. Red indicates high expression, and blue indicates low expression

Fig. 5 Tissue-specific expression analysis of GRAS genes in mulberry under salt stress* indicates significant difference at the P<0.05 level between the treatment group and the control group within the same group. The same below

Fig. 6 RT-qPCR detection of mulberry plants transiently transformed with MnGRAS22

Fig. 7 Salt-tolerance analysis of MnGRAS22 geneA: SOD activity; B: POD activity; C: CAT activity; D: MDA content; E: Proline content; F: DAB histochemical staining; G: NBT histochemical staining; H: Evans blue histochemical staining

Fig. 8 Expression analysis of antioxidant enzyme genes

References 51

[1]	Chen G, Li D, Yao P, et al. Metabolic and transcriptional analysis reveals flavonoid involvement in the drought stress response of mulberry leaves [J]. Int J Mol Sci, 2024, 25(13): 7417.
[2]	Pysh LD, Wysocka-Diller JW, Camilleri C, et al. The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes [J]. Plant J, 1999, 18(1): 111-119.
[3]	Hirsch S, Kim J, Muñoz A, et al. GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula [J]. Plant Cell, 2009, 21(2): 545-557.
[4]	Zhang H, Mi LM, Xu L, et al. Genome-wide identification, characterization, interaction network and expression profile of GRAS gene family in sweet orange (Citrus sinensis) [J]. Sci Rep, 2019, 9: 2156.
[5]	Li SP, Zhao YH, Zhao Z, et al. Crystal structure of the GRAS domain of SCARECROW-LIKE7 in Oryza sativa [J]. Plant Cell, 2016, 28(5): 1025-1034.
[6]	Sun XL, Jones WT, Harvey D, et al. N-terminal domains of DELLA proteins are intrinsically unstructured in the absence of interaction with GID1/gibberellic acid receptors [J]. J Biol Chem, 2010, 285(15): 11557-11571.
[7]	Gao MJ, Parkin I, Lydiate D, et al. An auxin-responsive SCARECROW-like transcriptional activator interacts with histone deacetylase [J]. Plant Mol Biol, 2004, 55(3): 417-431.
[8]	Wang Q, Yang YB, Yu YH, et al. Genome-wide characterization of the GRAS gene family in Cyclocarya paliurus and its involvement in heterodichogamy [J]. Agronomy, 2024, 14(10): 2397.
[9]	He ZH, Tian ZZ, Zhang Q, et al. Genome-wide identification, expression and salt stress tolerance analysis of the GRAS transcription factor family in Betula platyphylla [J]. Front Plant Sci, 2022, 13: 1022076.
[10]	Gao L, Shi AK, Wang M, et al. Genome-wide identification and expression profiling of the GRAS gene family in Taxus cuspidate [J]. Genes, 2025, 16(11): 1345.
[11]	毛可欣, 王海荣, 安淼, 等. 中华猕猴桃GRAS基因家族鉴定及低温胁迫表达分析 [J]. 生物技术通报, 2023, 39(11): 297-307.
	Mao KX, Wang HR, An M, et al. Identification of GRAS gene family and expression analysis under low temperature stress in Actinidia chinensis [J]. Biotechnol Bull, 2023, 39(11): 297-307.
[12]	Wu R, Liu WH, Liu KQ, et al. Genome-wide identification and expression of the GRAS gene family in oat (Avena sativa L.) [J]. Agronomy, 2023, 13(7): 1807.
[13]	Guan YY, Wang KG, Zhao JJ, et al. Genome-wide identification of TaeGRASs responsive to biotic stresses and functional analysis of TaeSCL6 in wheat resistance to powdery mildew [J]. BMC Genomics, 2024, 25(1): 1149.
[14]	Greb T, Clarenz O, Schafer E, et al. Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation [J]. Genes Dev, 2003, 17(9): 1175-1187.
[15]	Liu YD, Wen L, Shi Y, et al. Stress-responsive tomato gene SlGRAS4 function in drought stress and abscisic acid signaling [J]. Plant Sci, 2021, 304: 110804.
[16]	刁邓超, 李云丽, 孟祥宇, 等. 小麦TaGRAS34-5A的克隆及耐热功能研究 [J]. 中国农业科学, 2025, 58(4): 617-634.
	Diao DC, Li YL, Meng XY, et al. Cloning and heat tolerance function of wheat TaGRAS34-5A gene [J]. Sci Agric Sin, 2025, 58(4): 617-634.
[17]	Lin JT, Wu JH, Zhang D, et al. The GRAS gene family and its roles in pineapple (Ananas comosus L.) developmental regulation and cold tolerance [J]. BMC Plant Biol, 2024, 24(1): 1204.
[18]	Huang JY, Ren HF, Cheng F, et al. Comprehensive genome-wide characterization of the GRAS gene family and their role in salt stress tolerance in Punica granatum L. [J]. Horticulturae, 2025, 11(5): 504.
[19]	Ma HS, Liang D, Shuai P, et al. The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana [J]. J Exp Bot, 2010, 61(14): 4011-4019.
[20]	She M, Zheng DY, Zhang SP, et al. Functional analysis of maize GRAS transcription factor gene ZmGRAS72 in response to drought and salt stresses [J]. Agric Commun, 2024, 2(3): 100054.
[21]	Ni LJ, Wang ZQ, Liu XD, et al. Genome-wide study of the GRAS gene family in Hibiscus hamabo Sieb. et Zucc and analysis of HhGRAS14-induced drought and salt stress tolerance in Arabidopsis [J]. Plant Sci, 2022, 319: 111260.
[22]	Li WR, Yu JC, Wang RH, et al. Overexpression of SmGRAS5 enhances tolerance to abiotic stresses in Salvia miltiorrhiza [J]. Plant Physiol Biochem, 2025, 221: 109669.
[23]	Ma A, Wang TJ, Wang HR, et al. The GRAS transcription factor OsGRAS2 negatively impacts salt tolerance in rice [J]. Plant Cell Rep, 2024, 44(1): 17.
[24]	Khan Y, Xiong Z, Zhang H, et al. Expression and roles of GRAS gene family in plant growth, signal transduction, biotic and abiotic stress resistance and symbiosis formation—a review [J]. Plant Biol, 2022, 24(3): 404-416.
[25]	Dong XM, Han BC, Yin XY, et al. Genome-wide identification of the GRAS transcription factor family in autotetraploid cultivated alfalfa (Medicago sativa L.) and expression analysis under drought stress [J]. Ind Crops Prod, 2023, 194: 116379.
[26]	Hao XL, Gong YY, Chen SX, et al. Genome-wide identification of GRAS transcription factors and their functional analysis in salt stress response in sugar beet [J]. Int J Mol Sci, 2024, 25(13): 7132.
[27]	Sun XL, Xue B, Jones WT, et al. A functionally required unfoldome from the plant Kingdom: intrinsically disordered N-terminal domains of GRAS proteins are involved in molecular recognition during plant development [J]. Plant Mol Biol, 2011, 77(3): 205-223.
[28]	Lamesch P, Berardini TZ, Li DH, et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools [J]. Nucleic Acids Res, 2012, 40(Database issue): D1202-D1210.
[29]	Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020 [J]. Nucleic Acids Res, 2021, 49(D1): D458-D460.
[30]	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
[31]	Chou KC, Shen HB. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization [J]. PLoS One, 2010, 5(6): e11335.
[32]	Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol Biol Evol, 2016, 33(7): 1870-1874.
[33]	He ZL, Zhang HK, Gao SH, et al. Evolview v2: an online visualization and management tool for customized and annotated phylogenetic trees [J]. Nucleic Acids Res, 2016, 44(W1): W236-W241.
[34]	Wang JY, Chitsaz F, Derbyshire MK, et al. The conserved domain database in 2023 [J]. Nucleic Acids Res, 2023, 51(D1): D384-D388.
[35]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences [J]. Nucleic Acids Res, 2002, 30(1): 325-327.
[36]	董亚茹, 李茹霞, 赵东晓, 等. 7个桑树AP2/ERF转录因子的生物信息学及表达分析 [J]. 山东农业科学, 2022, 54(1): 7-13.
	Dong YR, Li RX, Zhao DX, et al. Bioinformatics and expression analyses of 7 mulberry AP2/ERF transcription factors [J]. Shandong Agric Sci, 2022, 54(1): 7-13.
[37]	Ji XY, Zheng L, Liu YJ, et al. A transient transformation system for the functional characterization of genes involved in stress response [J]. Plant Mol Biol Report, 2014, 32(3): 732-739.
[38]	陈建勋, 王晓峰. 植物生理学实验指导 [M]. 广州: 华南理工大学出版社, 2002.
	Chen JX, Wang XF. Experimental instruction of plant physiology [M]. Guangzhou: South China University of Technology Press, 2002.
[39]	Allen P, Bennett K, Heritage B. SPSS Statistics Version 22: A Practical Guide [M]. 3rd ed. Sydney: Cengage Learning Australia Pty Limited, 2014.
[40]	Corporation OriginLab. Origin 2024 [CP]. Northampton: OriginLab Corporation, 2023.
[41]	Inc Adobe. Adobe Illustrator 29.8 [CP]. San Jose: Adobe Inc., 2025.
[42]	Sun Y, Yuan T. Genome-wide analysis of GRAS gene family and functional identification of a putative development and maintenance of axillary meristematic tissue gene PlGRAS22 in Paeonia ludlowii [J]. Int J Biol Macromol, 2025, 297: 139879.
[43]	孔青洋, 张晓龙, 李娜, 等. 单叶蔷薇GRAS转录因子家族鉴定及表达分析 [J]. 生物技术通报, 2025, 41(1): 210-220.
	Kong QY, Zhang XL, Li N, et al. Identification and expression analysis of GRAS transcription factor family in Rosa persica [J]. Biotechnol Bull, 2025, 41(1): 210-220.
[44]	Chen Y, Zhu PP, Wu SY, et al. Identification and expression analysis of GRAS transcription factors in the wild relative of sweet potato Ipomoea trifida [J]. BMC Genomics, 2019, 20(1): 911.
[45]	Chen JW, Yan Q, Li JW, et al. The GRAS gene family and its roles in seed development in litchi (Litchi chinensis Sonn) [J]. BMC Plant Biol, 2021, 21(1): 423.
[46]	Ming RH, Fang T, Ling W, et al. The GRAS transcription factor PtrPAT1 of Poncirus trifoliata functions in cold tolerance and modulates glycine betaine content by regulating the BADH-like gene [J]. Hortic Res, 2024, 12(1): uhae296.
[47]	刘丽, 武琦, 高婷, 等. 太行菊GRAS基因家族鉴定及其在盐胁迫下的表达分析 [J]. 山西农业大学学报: 自然科学版, 2025, 45(6): 1-11.
	Liu L, Wu Q, Gao T, et al. Identification of the GRAS gene family in Opisthopappus taihangensis and its analysis expression under salt stress [J]. J Shanxi Agric Univ Nat Sci Ed, 2025, 45(6): 1-11.
[48]	Weng YH, Chen XY, Hao ZD, et al. Genome-wide analysis of the GRAS gene family in Liriodendron chinense reveals the putative function in abiotic stress and plant development [J]. Front Plant Sci, 2023, 14: 1211853.
[49]	Zentella R, Zhang ZL, Park M, et al. Global analysis of della direct targets in early gibberellin signaling in Arabidopsis [J]. Plant Cell, 2007, 19(10): 3037-3057.
[50]	Tong N, Li D, Zhang ST, et al. Genome-wide identification and expression analysis of the GRAS family under low-temperature stress in bananas [J]. Front Plant Sci, 2023, 14: 1216070.
[51]	Li L, Tian ZZ, Zhao ZZ, et al. The GRAS transcription factor BpGRAS34 functions as a positive regulator of salt stress response in Betula platyphylla [J]. Plant Sci, 2025, 358: 112575.