bHLH-regulated routes in anther development in rice and Arabidopsis

Ortolan, Francieli; Trenz, Thomaz Stumpf; Delaix, Camila Luiza; Lazzarotto, Fernanda; Margis-Pinheiro, Marcia

doi:10.1590/1678-4685-GMB-2023-0171

Francieli Ortolan

wrote the manuscript

designed and produced the figures

read and approved the final version

Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Porto Alegre, RS, Brazil.

https://orcid.org/0000-0001-9040-6503

Thomaz Stumpf Trenz

wrote the manuscript

designed and produced the figures

read and approved the final version

Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Porto Alegre, RS, Brazil.

Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brazil.

https://orcid.org/0000-0001-6900-7831

Camila Luiza Delaix

designed and produced the figures

read and approved the final version

Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brazil.

https://orcid.org/0000-0002-1765-8920

Fernanda Lazzarotto

revised and edited the manuscript

read and approved the final version

Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Porto Alegre, RS, Brazil.

Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brazil.

https://orcid.org/0000-0003-3257-5952

Marcia Margis-Pinheiro

revised and edited the manuscript

read and approved the final version

Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Porto Alegre, RS, Brazil.

Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Programa de Pós-Graduação em Biologia Celular e Molecular, Porto Alegre, RS, Brazil.

https://orcid.org/0000-0001-5356-3497

Send correspondence to Marcia Margis-Pinheiro. Universidade Federal do Rio Grande do Sul, Programa de Pós-graduação em Genética e Biologia Molecular, Departamento de Genética, Av. Bento Gonçalves, 9500, Agronomia, 91.501- 970, Porto Alegre, RS, Brazil. E-mail: marcia.margis@ufrgs.br.

Associate Editor:

Loreta Brandão de Freitas

Conflict of Interest

The authors declare that there is no conflict of interest

Figures (3)
Tables (1)

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Figure 1 -
The expression of TFs in the anther development in rice and Arabidopsis. Schematic representation of anther development classified into 14 stages in rice and Arabidopsis, based on Sanders et al. (1999Sanders PM, Bui AQ, Weterings K and McIntire KN (1999) Histochemical staining of Arabidopsis thaliana pollen tubes for β-glucuronidase activity. Sex Plant Reprod 12:322-329.), Itoh et al. (2005Itoh JI, Nonomura KI, Ikeda K, Yamaki S, Inukai Y, Yamagishi H, Kitano H and Nagato Y (2005) Rice plant development: From zygote to spikelet. Plant Cell Physiol 46:23-47.), and Zhang et al. (2011Zhang D, Luo X and Zhu L (2011) Cytological analysis and genetic control of rice anther development. J Gene t Genomics 38:379-390.). Phases in this representation highlight the primary events occurring in respective stages. The stages related to cell fate determination range from 1 to 5 in rice and 1 to 4 in Arabidopsis. The meiotic phase includes stages 6, 7, 8a, and 8b in rice, and stages 5, 6 and 7 in Arabidopsis. The microspore maturation phase spans stages 9 to 12 in rice and 8 to 12 in Arabidopsis. Finally, anther dehiscence takes place during stages 13 and 14 in both rice and Arabidopsis. The color of each gene is the same between orthologs in rice and Arabidopsis. L1, L2, L3: cell layers in stamen primordia; E: epidermis; 2°P: secondary parietal layers; 1°Sp: primary sporogenous cells; 2°Sp: secondary sporogenous cells; En: endothecium; ML: middle layer; T: tapetum; MMC: microspore mother cell; MC: meiotic cell; Dy: dyad cell. Tds: tetrads. Msp: microspore parietal cell. MP: mature pollen. GAMYB: GIBBERELLIN MYB GENE; UDT1: UNDEVELOPED TAPETUM1; TDF1: DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1; TIP2: bHLH142/TDR INTERACTING PROTEIN2; TIP3: TDR INTERACTING PROTEIN3; TDR: TAPETUM DEGENERATION RETARDATION; EAT1: ETERNAL TAPETUM1; MS1: MALE STERILITY1; DYT1: DYSFUNCTIONAL TAPETUM1; AMS: ABORTED MICROSPORES.

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Figure 2 -
Proposed model for bHLH regulatory pathway of anther development in rice. The top panel illustrates the regulatory network primarily active during the meiotic phase of anther development, with a predominant role of bHLH transcription factors. In contrast, the bottom panel depicts the regulatory pathway during the microspore maturation phase, highlighting genes directly associated with tapetum programmed cell death (PCD) and pollen wall formation. Lines indicate direct regulation, while dashed lines indicate possible regulation. Arrows indicate induction while dashes indicate repression. Taper lines indicate the product of the expression. The color of the boxes in the legend, as well as the color and shape of the protein are the same between the orthologs in Arabidopsis in . UDT1: UNDEVELOPED TAPETUM1; TDF1: DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1; TIP2: bHLH142/TDR INTERACTING PROTEIN2; GAMYB: GIBBERELLIN MYB GENE; TDR: TAPETUM DEGENERATION RETARDATION; EAT1: ETERNAL TAPETUM1; OsCP1: CYSTEINE PROTEASE1; OsADF: ANTHER DEVELOPMENT F-BOX; OsC6: CYSTEINE PROTEASE6; TIP3: TDR INTERACTING PROTEIN3; OsMS1: MALE STERILITY1; OsCYP703A3/OsCYP704B2: CYTOCHROME P450 MEMBERS; OsPKS1: POLYKETIDE SYNTHASE1; OsPKS2: POLYKETIDE SYNTHASE2; OsDPW: DEFECTIVE POLLEN WALL; OsABCG15: ABC TRANSPORTER G15; OsAP25: ASPARTIC PROTEASE25; OsAP37: ASPARTIC PROTEASE37; OsLTPL94: NON-SPECIFIC LIPID TRANSFER PROTEIN94; 24-PHAS: 24-NUCLEOTIDE PHASED SECONDARY SMALL INTERFERING RNAS; DCL5: DICER-LIKE5.

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Figure 3 -
Arabidopsis bHLH involvement in molecular mechanisms of anther development. A) DYT1 is localized in the cytoplasm and nucleus compartments at anther stage 5. It induces the expression of other bHLH genes, whose products interact with DYT1, resulting in DYT1-bHLH010/089/091 heterodimers at anther stage 6. These heterodimers facilitate DYT1 nuclear localization, and their different interaction combinations trigger the transcription of downstream genes, such as TDF1, EXL4, and ATA20. The accumulation of DYT1-bHLH010/089/091 heterodimers suppresses directly or indirectly the expression of DYT1 by negative feedback. B) AMS role during outer pollen layer (exine) wall formation. DYT1 binds to the promoter of the TDF1 transcription factor and activates its expression. TDF1 activates the expression of downstream genes, such as AMS. AMS binds to the promoter of genes related to fatty acid elongation (KCS15), fatty acid hydroxylation (CYP98A8), lipid metabolism/transport (LTP), and production of hydroxylated 𝛂-pyrones (TKPR1, PKSB), which products are involved in the synthesis of the precursors of sporopollenin biopolymer, to form the exine. Also, AMS potentially activates the expression of pollen coat proteins (GRP18, GRP19, EXL5, EXL6) by binding their promoters and can form heterodimers with TDF1 to trigger the expression of MYB103 and TEK. MYB103 directly activates the expression of MS1, which also activates the expression of pollen coat proteins. C) Arabidopsis bHLHs feedback regulations. BES1 binds to the DYT1 promoter and might activate its expression. DYT1 interacts with other bHLHs and regulates several genes, including TDF1. TDF1 activates the expression of AMS, which product can also interact with many bHLH and regulate a plethora of genes. AMS can promote the expression of MYB103, which activates the transcription of MS1. MS1 represses the expression of TDF1 and decreases the AMS protein levels and gene expression, potentially by protein degradation and chromatin remodeling, respectively. AMS interacts with the DYT1 protein and can regulate its expression. The competitive interaction among different bHLHs and transcription factors results in a controlled and coordinated activation/repression of genes involved in anther development and pollen formation. Lines ending with an arrow: direct regulation. Lines ending with a line: repression. Dashed lines: Directly/indirectly or unknown. Red lines: regulation of AMS via MS1. The color of the boxes in the legend, as well as the color and shape of the protein are the same between the orthologs in rice in . DYT1: DYSFUNCTIONAL TAPETUM1; EXL4/5/6: EXTRACELLULAR LIPASE 4/5/6; ATA20: ANTHER 20; AMS: ABORTED MICROSPORES; TDF1: DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1; BES1: BRI1 EMS SUPPRESSOR 1; KCS15: 3-KETOACYL-COA SYNTHASE 15; CYP98A8/9: CYTOCHROME P450, FAMILY 98, SUBFAMILY A, POLYPEPTIDE 8/9; TKPR1: TETRAKETIDE 𝛂-PYRONE REDUCTASE1; PKSB: POLYKETIDE SYNTHASE B; GRP18/19: GLYCINE-RICH PROTEIN 18/19; MS1: MALE STERILITY1.

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Table 1 -
bHLHs involved in anther development

Figure 3 - Arabidopsis bHLH involvement in molecular mechanisms of anther development. A) DYT1 is localized in the cytoplasm and nucleus compartments at anther stage 5. It induces the expression of other bHLH genes, whose products interact with DYT1, resulting in DYT1-bHLH010/089/091 heterodimers at anther stage 6. These heterodimers facilitate DYT1 nuclear localization, and their different interaction combinations trigger the transcription of downstream genes, such as TDF1, EXL4, and ATA20. The accumulation of DYT1-bHLH010/089/091 heterodimers suppresses directly or indirectly the expression of DYT1 by negative feedback. B) AMS role during outer pollen layer (exine) wall formation. DYT1 binds to the promoter of the TDF1 transcription factor and activates its expression. TDF1 activates the expression of downstream genes, such as AMS. AMS binds to the promoter of genes related to fatty acid elongation (KCS15), fatty acid hydroxylation (CYP98A8), lipid metabolism/transport (LTP), and production of hydroxylated 𝛂-pyrones (TKPR1, PKSB), which products are involved in the synthesis of the precursors of sporopollenin biopolymer, to form the exine. Also, AMS potentially activates the expression of pollen coat proteins (GRP18, GRP19, EXL5, EXL6) by binding their promoters and can form heterodimers with TDF1 to trigger the expression of MYB103 and TEK. MYB103 directly activates the expression of MS1, which also activates the expression of pollen coat proteins. C) Arabidopsis bHLHs feedback regulations. BES1 binds to the DYT1 promoter and might activate its expression. DYT1 interacts with other bHLHs and regulates several genes, including TDF1. TDF1 activates the expression of AMS, which product can also interact with many bHLH and regulate a plethora of genes. AMS can promote the expression of MYB103, which activates the transcription of MS1. MS1 represses the expression of TDF1 and decreases the AMS protein levels and gene expression, potentially by protein degradation and chromatin remodeling, respectively. AMS interacts with the DYT1 protein and can regulate its expression. The competitive interaction among different bHLHs and transcription factors results in a controlled and coordinated activation/repression of genes involved in anther development and pollen formation. Lines ending with an arrow: direct regulation. Lines ending with a line: repression. Dashed lines: Directly/indirectly or unknown. Red lines: regulation of AMS via MS1. The color of the boxes in the legend, as well as the color and shape of the protein are the same between the orthologs in rice in Figure 2. DYT1: DYSFUNCTIONAL TAPETUM1; EXL4/5/6: EXTRACELLULAR LIPASE 4/5/6; ATA20: ANTHER 20; AMS: ABORTED MICROSPORES; TDF1: DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1; BES1: BRI1 EMS SUPPRESSOR 1; KCS15: 3-KETOACYL-COA SYNTHASE 15; CYP98A8/9: CYTOCHROME P450, FAMILY 98, SUBFAMILY A, POLYPEPTIDE 8/9; TKPR1: TETRAKETIDE 𝛂-PYRONE REDUCTASE1; PKSB: POLYKETIDE SYNTHASE B; GRP18/19: GLYCINE-RICH PROTEIN 18/19; MS1: MALE STERILITY1.

Gene	Locus	Mutant phenotype	References
UDT1, UNDEVELOPED TAPETUM 1 (OsbHLH164)	LOC_Os07g36460	The udt1 mutant resulted in male sterility due to abnormal tapetal development and inhibited meiocyte development	Jung et al., 2005Jung KH, Han MJ, Lee YS, Kim YW, Hwang I, Kim MJ, Kim YK, Nahm BK and An G (2005) Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17:2705-2722..
DYT1, DYSFUNCTIONAL TAPETUM 1	At4g21330	The dyt mutant shows abnormal anther morphology, and meiocytes can complete meiosis I but do not form a thick callose wall, frequently fail to complete meiotic cytokinesis, and eventually collapse.	Cui et al., 2016Cui J, You C, Zhu E, Huang Q, Ma H and Chang F (2016) Feedback regulation of DYT1 by interactions with downstream bHLH factors promotes DYT1 nuclear plant jlocalization and anther development. Plant Cell 28:1078-1093.. Zhang et al., 2006Zhang W, Sun Y, Timofejeva L, Chen C, Grossniklaus U and Ma H (2006) Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor. Development 133:3085-3095..
TDR, TAPETUM DEGENERATION RETARDATION (OsbHLH005)	LOC_Os02g02820	The tdr mutant is male sterile and shows delayed degeneration of the tapetum and middle layer, along with the collapse of microspores.	Li et al., 2006Li N, Zhang DS, Liu HS, Yin CS, Li XX, Liang WQ, Yung Z, Xu B, Chu HW, Wang J et al. (2006) The rice Tapetum Degeneration Retardation gene is required for tapetum degradation and anther development. Plant Cell 18:2999-3014..
AMS, ABORTED MICROSPORE	At2g16910	The ams mutants displayed impaired release of microspores, a deficiency in sporopollenin deposition, and a substantial decrease in total phenolic compounds and cutin monomers.	Xu et al., 2014Xu J, Ding Z, Vizcay-Barrena G, Shi J, Liang W, Yuan Z, Werk-Reichhart D, Schreiber L, Wilson ZA and Zhang D (2014) ABORTED MICROSPORES acts as a master regulator of pollen wall formation in Arabidopsis. Plant Cell 26:1544-1556.. Sorensen et al., 2003Sorensen AM, Kröber S, Unte US, Huijser P, Dekker K and Saedler H (2003) The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor. Plant J 33:413-423..
TIP2, TDR INTERACTING PROTEIN 2 (OsbHLH142)	LOC_Os01g18870	The tip2 mutants manifest complete male sterility as they exhibit undifferentiated inner three layers of anther wall and fail to undergo tapetal programmed cell death.	Ko et al., 2017Ko SS, Li MJ, Lin YJ, Hsing HX, Yang TT, Chen TK, Jhong CM and Ku MSB (2017) Tightly controlled expression of bHLH142 is essential for timely tapetal programmed cell death and pollen development in rice. Front Plant Sci 8:1258.. Fu et al., 2014Fu Z, Yu J, Cheng X, Zong X, Xu J, Chen M, Li Z, Zhang D and Liang W (2014) The rice basic helix-loop-helix transcription factor TDR INTERACTING PROTEIN2 is a central switch in early anther development. Plant Cell 26:1512-1524..
bHLH010 bHLH089 bHLH091	At2g31220 At1g06170 At2g31210	The combinations of double and triple mutations exhibit strong anther phenotypes such as abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development.	Zhu et al., 2015Zhu E, You C, Wang S, Cui J, Niu B, Wang Y, Qi J, Ma H and Chang F (2015) The DYT 1‐interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome. Plant J 83:976-990..
EAT1, ETERNAL TAPETUM 1 (OsbHLH141)	LOC_Os04g51070	The eat1 mutant displays delayed meiosis with abnormally decondensed chromosomes, leading to the formation of abortive microspores due to abnormal programmed cell death of the tapetum.	Niu et al., 2013Niu N, Liang W, Yang X, Jin W, Wilson ZA, Hu J and Zhang D (2013) EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat Commun 4:1445..
OsbHLH035	LOC_Os01g06640	OsbHLH035 overexpression plants displayed smaller and curved anthers. The anthers exhibited abnormal changes in the endothecium, sterile pollen sacs, and pollen grains with atypical vacuolation, as well as changes in size.	Ortolan et al., 2021Ortolan F, Fonini LS, Pastori T, Mariath JE, Saibo NJ, Margis-Pinheiro M and Lazzarotto F (2021) Tightly controlled expression of OsbHLH35 is critical for anther development in rice. Plant Sci 302:110716..

[1] Send correspondence to Marcia Margis-Pinheiro. Universidade Federal do Rio Grande do Sul, Programa de Pós-graduação em Genética e Biologia Molecular, Departamento de Genética, Av. Bento Gonçalves, 9500, Agronomia, 91.501- 970, Porto Alegre, RS, Brazil. E-mail: marcia.margis@ufrgs.br.

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bHLH-regulated routes in anther development in rice and Arabidopsis