Genome-Wide Identification and Analysis of bZIP Transcription Factor Gene Family in Broomcorn Millet (Panicum miliaceum L.)

Abstract

Background: Basic (region) leucine zippers (bZIPs) make up one of the largest families and are some of the most prevalent evolutionarily conserved transcription factors (TFs) in eukaryotic organisms. Plant bZIP family members are involved in seed germination, vegetative growth, flower development, light response, and various biotic/abiotic stress response pathways. Nevertheless, a detailed identification and genome-wide analysis of the bZIP family genes in broomcorn millet have not been conducted. Methods: In this research, we performed genome-wide identification, phylogenetic analysis, cis-elements analysis, and expression pattern analysis. Results: 144 bZIP transcription factors were identified from the P. miliaceum genome and classified into eleven subfamilies using a phylogenetic analysis. Motif and bZIP domain sequence alignment analyses indicated that the members in each subfamily were relatively conserved. Furthermore, a promoter analysis revealed that bZIP transcription factor family genes were responsive to multiple hormones and environmental stresses. Additionally, cis-element MYB binding sites were identified in the promoters of most PmbZIP genes. A gene expression analysis showed that 18 PmbZIP genes were differentially expressed during seed germination in salt stress, with 7 being significantly downregulated and 11 upregulated, thus suggesting that these PmbZIP genes may play an important role in the salt stress response and seed germination. Conclusions: Current research provides valuable information for further functional analyses of the PmbZIP gene family and as a reference for future studies on broomcorn millet’s stress response.

1. Introduction

Broomcorn millet (Panicum miliaceum L.) is one of the plants that was cultivated the earliest in the world and is also known as millet, common millet, or proso millet. It has been cultivated for more than 10,000 years and played significant roles in food security and cultural history in China [1]. Broomcorn millet has a short growing period and is the most water-efficient; has a high salt tolerance and good nutrient resource usage efficiency; and is high in proteins, certain minerals, and antioxidants compared to most other cereals [2,3,4]. Broomcorn millet is an allotetraploid with 36 chromosomes (2n = 4× = 36) [5], and its genome size is approximated to be ~923 Mb [6]. As a C4 photosynthesis crop that is closely related to the bioenergy crop switchgrass (Panicum virgatum L.), broomcorn millet has been reported to have the maximum water use efficiency (WUE), which may be due to its slow breathing rate, short generation time (about 60–90 days), and high harvest index [7,8,9]. Progress in molecular biology research on broomcorn millet is slow, and only a simple genetic map and a few genetic markers have been found. In 2019, the millet genome was sequenced, and phylogenetic analysis showed that millet divided into broomcorn millet and foxtail millet ~13.1 million years ago (Mya), while broomcorn millet became an allotetraploid ~5.91 million years ago [10]. One study also identified C4 candidate genes in broomcorn millet, and these genes were found to be spread over all three C4 subtypes, thus suggesting that broomcorn millet coexisted in three different carbon fixation pathways [6]. In the immediate future, broomcorn millet will become a crucial crop that can help in diversifying agriculture and promoting a healthier diet for humans. Therefore, searching for and identifying advantageous genes in millet will facilitate further basic scientific research on molecular breeding processes.

Transcription factors (TFs, also called trans-regulatory factors) are proteins with DNA-binding function, and they bind to specific cis-regulatory elements (cis-elements) and directly regulate the transcription of DNA to mRNA [11]. There are more divergent TF families and more unique DNA-binding domains (DBDs) in plant genomes [12], which also play a vital role in the regulation of growth, development, and environmental response. Structurally, TFs are usually classified by their DNA-binding domains: Basic (region) leucine zipper (bZIP) TFs have a basic region that binds DNA in the N-terminal and a leucine zipper dimerization motif in the C-terminal [13]. The basic DNA-binding region is an invariant N-X7-R/K motif, which has asparagine (N) and basic (R/K) residues with exact spacing; meanwhile, the ZIP domain within an alpha helix consists of heptad repeats of leucine (L) or a related hydrophobic amino acid [14]. Charged amino acids produce attractive and repulsive g-e’ pairs to regulate dimerization specificity and bind to DNA [15].

Among various transcription factor families in plants, basic leucine zipper (bZIP) proteins represent one of the most conserved and extensively studied groups. They are involved in diverse biological processes, particularly in mediating plant responses to abiotic stresses such as drought, salinity, and cold. bZIP transcription factors are well known for their central role in abscisic acid (ABA)-dependent signaling pathways, especially the PP2C–SnRK2–AREB cascade, which regulates key stress-adaptive responses and seed germination [16]. Compared to other transcription factor families (e.g., zinc finger and bHLH), bZIP genes have been more directly linked to core hormonal and environmental response mechanisms. To date, many plant bZIP transcription factor families have been identified and characterized extensively, including 78 AtbZIPs in Arabidopsis thaliana [13,14], 89 OsbZIPs in rice (Oryza sativa) [17], 187 TabZIPs in wheat (Triticum aestivum) [18], 85 SibZIPs in foxtail millet (Setaria italica), 103 OebZIPs in olive (Olea europaea) [19], 154 PhebZIPs in bamboo (Phyllostachys edulis) [20], 247 BnbZIPs in Brassica napus [21], 50 FvbZIPs in strawberry (Fragaria vesca) [22], 115 ZlbZIPs in Zizania latifolia [23], and 66 ItfbZIPs in sweet potato (Ipomoea trifida) [24]. Numerous studies have shown that different subgroups of bZIP TFs are involved in multiple regulation pathways in plants. bZIP TFs in Group A mainly take part in ABA signaling [25,26,27], abiotic stress responses [28,29,30,31], seed germination [32,33], and plant floral transition control [34,35,36]; bZIP TFs in Groups B and K are important regulators of endoplasmic reticulum (ER) stress response [14,37]; those in Groups C and S regulate sucrose signaling and seed storage protein production [38,39,40]; those in Group D are involved in detoxification processes and pathogen defense responses with salicylic acid (SA) defenses against biotrophic pathogens, as well as the defense of hormones jasmonic acid (JA) and ethylene (ET) against necrotrophies [14,41,42,43,44,45,46]; E-members and M-members might control pollen development [47]; F subfamily members control genes that encode for Zn transporters [14,48,49]; those in Groups G and J have been reported to regulate ER–Golgi transport and pathogen defense [50,51]; H-members are implicated in multiple hormone signaling pathways and development during light regulation [52,53,54]; bZIP TFs in Group I participate in osmosensory responses and root bending regulation [55,56]. bZIP target sequences often contain an ACGT core [57], such as G-box (CACGTG), C-box (GACGTC), A-box (TACGTA), etc. However, one investigator also found that some bZIPs can bind non-ACGT sequences [58,59].

Although the function of bZIP TFs has been reported and their identification and analysis have been conducted in Arabidopsis and many species, their roles in broomcorn millet remain largely unknown. In our previous transcriptome analysis of broomcorn millet (P.miliaceum) under salt stress [60], we observed that a substantial number of bZIP genes were differentially expressed during seed germination. This suggests that bZIP transcription factors may play important regulatory roles in the stress tolerance mechanisms of this highly resilient cereal crop. This study systematically analyzes biological information about bZIP TFs in broomcorn millet, aiming to provide a reference for the identification of various functions.

2. Materials and Methods

2.1. Genome-Wide Identification and Prediction of Physicochemical Properties

Broomcorn millet genomic sequences, coding region sequences (coding sequence, CDS), and protein sequences were downloaded from NCBI under BioProject number PRJNA431363 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA431363, accessed on 23 June 2022). We referred to the Pfam number (PF00170) of bZIP transcription factors in the Pfam database (http://pfam.xfam.org/, accessed on 23 June 2022) and used the HMMER program based on a Hidden Markov Model (3.3.2) to search for the candidate bZIP proteins in broomcorn millet. The HMM profile was used to perform an hmmscan search against the P.miliaceum protein database with an E-value threshold of 1 × 10⁻⁵. We used protein sequences with a result greater than “0” as candidate sequences, and then, the candidate gene protein sequence was extracted using TBtools (v1.0692) [61]. Finally, all candidate protein sequences were further detected and identified via CDD (http://www.ncbi.nlm.nih.gov/cdd/, accessed on 22 June 2022) and PFAM, and proteins without bZIP domains were removed. The proteins were named according to the location of the bZIP transcription factor on the chromosome. The theoretical isoelectric point and molecular weight of the bZIP transcription factor proteins in broomcorn millet were calculated using ExPASy—Compute pI/Mw (https://web.expasy.org/compute_pi/, accessed on 25 June 2022).

2.2. Phylogeny Analyses and bZIP Domain Amino Acid Sequence Alignment

A.thaliana bZIP TF protein sequences were downloaded from the database TAIR (https://www.arabidopsis.org/index.jsp, accessed on 2 July 2022), and S.italica bZIP TF protein sequences were downloaded from the database Phytozome v12 (http://phytozome.jgi.doe.gov/pz/portal.html, accessed on 2 July 2022). A bootstrap neighbor-joining (NJ) evolutionary tree was created using MEGA 6.06 (https://www.megasoftware.net/, accessed on 7 July 2022) software with 1000 bootstrap replicates based on the sequence alignments. Additionally, the sequence alignment of the bZIP domain from broomcorn millet was performed using Clustal X 1.8.

2.3. Motif and Intron/Exon Gene Structure Analysis

The MEME v5.3.0 online service (http://meme-suite.org/tools/meme, accessed on 13 August 2022) was employed to identify conserved motifs in PmbZIP proteins, using parameters that included a maximum of 12 motifs and an optimal motif width ranging from 6 to 50 amino acids. Afterward, all identified motifs were annotated using InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/, accessed on 16 August 2022), and the gene structure display server program (http://gsds.cbi.pku.edu.cn/, accessed on 30 August 2022) was used to draw the gene structures of PmbZIPs.

2.4. Promoter Analysis

The 2000 base pairs preceding the initiation codon of each PmbZIP gene was obtained. These sequences were analyzed to find cis-elements using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 October 2022), and the outcomes were visualized with TBtools (v1.0692) [61].

2.5. RNA Isolation and bZIP Gene Expression Analysis

The samples of yumi1 and yumi9, which were grown in 0 mM of NaCl (RO water) and 250 mM NaCl, respectively, were collected at 0 h and 3 h, and dew white seeds were harvested after continuous light conditions commenced. (There were three independent biological replicates for each sample). The samples were swiftly frozen with liquid nitrogen and stored at −80 °C. In total, thirty-six samples were employed for RNA-Seq and differential expression analyses.

For transcriptome analysis, total RNA was isolated from seeds using the RNA prep Pure polysaccharide polyphenol plant total RNA extraction kit (DP441) (TIANGEN, Beijing, China). Using 1% agarose gels tested RNA degradation and contamination, and RNA purity was evaluated using a NanoPhotometer^® spectrophotometer (IMPLEN, Munich, Germany). RNA concentration was measured using Qubit^® RNA Assay Kit in Qubit^® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). The Agilent Bioanalyzer 2100 system’s RNA Nano 6000 Assay Kit was used to assess RNA integrity (Agilent Technologies, Santa Clara, CA, USA).

A differential expression analysis of two conditions/groups was performed. The DESeq R package (version 1.10.1) was used to analyze differential expression between two conditions or groups. DESeq offers statistical methods to identify differential expression data, utilizing a model based on the negative binomial distribution. The Benjamini and Hochberg method was used to adjust the resulting p-values to control the false discovery rate, and differentially expressed genes were those with an adjusted p-value under 0.05. The creation of heatmaps and the cluster analysis of PmbZIPs were accomplished using TBtools software [61].

3. Results

3.1. Identification of PmbZIPs in Broomcorn Millet

To identify bZIP genes in the complete P. miliaceum genome, the Hidden Markov Model (HMM) profile file of the bZIP domain (PF00170) was exploited as a query file for a search across the P. miliaceum protein sequence data, and the Pfam and CDD databases were used to confirm the presence of the complete bZIP domain. As shown in Table 1, we identified 144 PmbZIP genes in the P. miliaceum genome after removing redundant sequences and designated them as PmbZIP1 to PmbZIP144 according to their chromosome locus. Moreover, the physical and chemical properties of the 144 PmbZIPs were analyzed, such as amino acid (aa) length, molecular weight (MW), and protein isoelectric (PI) points. Chromosomal localization shows that there are 14 PmbZIPs in Chr5, which has the most bZIP genes, but Chr15, Chr16, and Chr17 only have 2 PmbZIPs. Genome sequence analyses were conducted and showed that PmbZIPs ranged from 369 base pairs (bp, PmbZIP100, PmbZIP114) to 12,828 bp (PmbZIP45). Protein sequence analyses showed that the PmbZIPs ranged from 78 aa (PmbZIP59) to 650 aa (PmbZIP51) in length. The predicted MWs ranged from 9.19 kDa (PmbZIP59) to 68.3 kDa (PmbZIP51), and the PIs ranged from 4.52 (PmbZIP102) to 11.57 (PmbZIP43).

Table 1.

Identification and summary of information on Panicum miliaceum bZIP transcription factors (PmbZIPs) in broomcorn millet.

Gene ID	Protein Number	Gene Number	Chr	Gene Location	Gene Length/bp	CDS/nt	Amino Acids	PI	Molecular Weight/u
PmbZIP1	RLN41431.1	PM01G00370	1	454801–457374	2574	1263	421	5.71	44,828.00
PmbZIP2	RLN41707.1	PM01G08790	1	7046270–7059039	12,770	1149	383	9.13	40,969.23
PmbZIP3	RLN40362.1	PM01G12600	1	10097025–10098402	1378	486	162	10.55	17,692.55
PmbZIP4	RLN40088.1	PM01G13370	1	10863218–10866321	3104	912	304	9.45	34,047.74
PmbZIP5	RLN40734.1	PM01G13620	1	11058035–11060820	2786	684	228	9.84	24,396.40
PmbZIP6	RLN39254.1	PM01G14300	1	11670497–11674442	3946	1053	351	6.23	37,904.15
PmbZIP7	RLN41556.1	PM01G19740	1	16954898–16957707	2810	1161	387	7.23	41,543.20
PmbZIP8	RLN41102.1	PM01G22630	1	19957313–19960138	2826	1056	352	6.88	37,179.95
PmbZIP9	RLN42614.1	PM01G23610	1	20646169–20649807	3639	1242	414	5.06	42,598.17
PmbZIP10	RLN40049.1	PM01G26880	1	22949190–22950304	1115	615	205	10.18	22,321.56
PmbZIP11	RLN19317.1	PM02G26900	2	39030059–39034459	4401	1050	350	6.28	37,679.93
PmbZIP12	RLN19126.1	PM02G27550	2	39595051–39597823	2773	702	234	10.00	24,691.66
PmbZIP13	RLN18095.1	PM02G27800	2	39766627–39771830	5204	999	333	7.04	37,066.87
PmbZIP14	RLN17017.1	PM02G32070	2	43256944–43264901	7958	1149	383	9.20	40,881.04
PmbZIP15	RLN15790.1	PM02G38970	2	48295652–48298282	2631	1224	408	5.60	43,833.94
PmbZIP16	RLN15762.1	PM02G44000	2	51665787–51668165	2379	1161	387	6.10	41,310.03
PmbZIP17	RLN17495.1	PM02G44930	2	52341406–52345126	3721	1260	420	4.97	43,404.97
PmbZIP18	RLN18647.1	PM02G46570	2	53532299–53532877	579	579	193	10.48	21,390.51
PmbZIP19	RLN33487.1	PM03G00660	3	678937–679488	552	552	184	7.21	20,564.99
PmbZIP20	RLN33484.1	PM03G01530	3	1468835–1474450	5616	1014	338	9.88	36,231.59
PmbZIP21	RLN33244.1	PM03G03170	3	2666045–2670259	4215	1140	380	6.22	40,613.30
PmbZIP22	RLN32939.1	PM03G03540	3	2944837–2947710	2874	1464	488	9.20	51,669.75
PmbZIP23	RLN34898.1	PM03G04810	3	3889634–3890848	1215	639	213	6.84	22,223.95
PmbZIP24	RLN35613.1	PM03G16340	3	12865234–12869532	4299	1107	369	6.61	39,460.24
PmbZIP25	RLN33050.1	PM03G16790	3	13287052–13290598	3547	1005	335	8.90	37,308.18
PmbZIP26	RLN35551.1	PM03G16900	3	13342155–13343818	1664	783	261	10.02	28,735.61
PmbZIP27	RLN35711.1	PM03G21650	3	18768221–18771281	3061	1179	393	5.01	42,558.22
PmbZIP28	RLN33934.1	PM03G24730	3	22107028–22110718	3691	1161	387	6.61	39,600.43
PmbZIP29	RLN35629.1	PM03G29190	3	35167059–35175866	8808	1257	419	8.44	46,181.77
PmbZIP30	RLN35964.1	PM03G36770	3	57531413–57534679	3267	1122	374	5.10	39,436.23
PmbZIP31	RLM84895.1	PM04G07270	4	6860114–6863550	3437	1233	411	7.18	42,128.33
PmbZIP32	RLM86983.1	PM04G16890	4	29725581–29732074	6494	1122	374	9.54	41,241.35
PmbZIP33	RLM85912.1	PM04G17970	4	31027628–31030792	3165	1392	464	9.15	49,993.48
PmbZIP34	RLM85616.1	PM04G18960	4	31857501–31858542	1042	534	178	8.95	20,269.87
PmbZIP35	RLM87231.1	PM04G19820	4	32472417–32480988	8572	1491	497	7.51	55,166.25
PmbZIP36	RLM85816.1	PM04G21680	4	33883522–33886493	2972	1464	488	9.53	51,605.73
PmbZIP37	RLM86211.1	PM04G22100	4	34132892–34136810	3919	1140	380	6.38	40,629.39
PmbZIP38	RLM85470.1	PM04G22810	4	34716923–34718185	1263	753	251	7.83	26,101.09
PmbZIP39	RLM87366.1	PM04G29540	4	39691716–39694242	2527	1491	497	5.86	60,644.54
PmbZIP40	RLM85147.1	PM04G34030	4	42747464–42752113	4650	1116	372	6.27	39,659.45
PmbZIP41	RLM86171.1	PM04G34490	4	43064867–43067475	2609	1014	338	8.90	37,565.47
PmbZIP42	RLM87099.1	PM04G34620	4	43157454–43160933	3480	885	295	9.59	32,209.76
PmbZIP43	RLN30372.1	PM05G00470	5	481032–481791	760	495	165	11.57	17,411.98
PmbZIP44	RLN29606.1	PM05G02460	5	2429710–2436988	7279	1305	435	5.93	47,002.20
PmbZIP45	RLN28633.1	PM05G04900	5	4731398–4744225	12,828	1812	604	4.88	65,083.35
PmbZIP46	RLN27945.1	PM05G05230	5	5015435–5016619	1185	603	201	9.19	21,729.48
PmbZIP47	RLN28345.1	PM05G05380	5	5101092–5106900	5809	1449	483	6.25	52,474.70
PmbZIP48	RLN27488.1	PM05G08030	5	7121364–7128050	6687	1158	386	7.17	40,824.69
PmbZIP49	RLN29878.1	PM05G11380	5	10166559–10169754	3196	438	146	9.49	16,063.02
PmbZIP50	RLN30397.1	PM05G14850	5	13174076–13175991	1916	453	151	6.32	16,585.54
PmbZIP51	RLN29599.1	PM05G25250	5	46340685–46343488	2804	1950	650	8.15	68,282.36
PmbZIP52	RLN31068.1	PM05G29120	5	49501420–49504193	2774	1152	384	9.07	41,007.77
PmbZIP53	RLN28247.1	PM05G33670	5	53089874–53091765	1892	543	181	9.75	20,078.72
PmbZIP54	RLN28944.1	PM05G33910	5	53238531–53239814	1284	933	311	6.47	33,380.34
PmbZIP55	RLN28901.1	PM05G36440	5	55255118–55257485	2368	1062	354	6.68	39,751.11
PmbZIP56	RLN27558.1	PM05G36770	5	55582519–55584338	1820	780	260	9.55	26,952.16
PmbZIP57	RLN01174.1	PM06G02700	6	1946640–1949819	3180	1287	429	9.30	45,780.45
PmbZIP58	RLN00773.1	PM06G08160	6	5838864–5840465	1602	438	146	9.93	15,983.02
PmbZIP59	RLN01167.1	PM06G14080	6	10158766–10159216	451	234	78	10.63	9190.60
PmbZIP60	RLM98944.1	PM06G14410	6	10441629–10450559	8931	999	333	6.26	35,600.39
PmbZIP61	RLM98085.1	PM06G17810	6	12976476–12980172	3697	999	333	6.55	36,500.10
PmbZIP62	RLM98033.1	PM06G17990	6	13102719–13104001	1283	921	307	6.46	32,832.58
PmbZIP63	RLN00565.1	PM06G20460	6	14965993–14968992	3000	777	259	9.73	26,937.16
PmbZIP64	RLM99485.1	PM06G21690	6	16055800–16057296	1497	1497	499	7.03	52,598.69
PmbZIP65	RLM99017.1	PM06G30890	6	39341652–39342104	453	453	151	6.85	16,674.63
PmbZIP66	RLM99272.1	PM06G35160	6	43290439–43290954	516	516	172	11.51	18,029.63
PmbZIP67	RLN24624.1	PM07G00440	7	317276–318073	798	570	190	9.98	20,307.29
PmbZIP68	RLN25705.1	PM07G06850	7	6745210–6748914	3705	1080	360	7.69	40,074.24
PmbZIP69	RLN24094.1	PM07G10230	7	8252890–8253306	417	417	139	7.65	15,279.28
PmbZIP70	RLN24562.1	PM07G17750	7	36088695–36092402	3708	1110	370	9.19	39,245.04
PmbZIP71	RLN22304.1	PM07G23470	7	41273483–41276065	2583	804	268	5.93	28,650.69
PmbZIP72	RLN22621.1	PM07G25750	7	43086731–43089115	2385	981	327	7.18	35,585.99
PmbZIP73	RLN22673.1	PM07G29370	7	45806587–45808150	1564	1206	402	6.29	42,396.98
PmbZIP74	RLN23257.1	PM07G29380	7	45812241–45821193	8953	1227	409	6.90	46,071.37
PmbZIP75	RLN24712.1	PM07G29810	7	46066329–46066976	648	648	216	9.75	21,645.44
PmbZIP76	RLN25012.1	PM07G36760	7	51457907–51459054	1148	564	188	10.01	20,446.76
PmbZIP77	RLN24441.1	PM07G38580	7	53023815–53026143	2329	921	307	6.62	32,963.72
PmbZIP78	RLM93109.1	PM08G01760	8	1448785–1451902	3118	999	333	6.00	36,038.19
PmbZIP79	RLM93604.1	PM08G03930	8	3296164–3296959	796	585	195	9.94	20,689.71
PmbZIP80	RLM91692.1	PM08G15750	8	30944899–30945294	396	396	132	8.40	14,577.57
PmbZIP81	RLM92180.1	PM08G20990	8	36451978–36454357	2380	831	277	9.31	30,542.73
PmbZIP82	RLM93703.1	PM08G25890	8	40248925–40252095	3171	819	273	6.01	28,971.03
PmbZIP83	RLM92419.1	PM08G28810	8	42140458–42146113	5656	1401	467	6.34	50,908.00
PmbZIP84	RLM94116.1	PM08G29030	8	42303295–42305878	2584	981	327	7.15	35,542.86
PmbZIP85	RLN12842.1	PM09G06500	9	5334910–5339163	4254	1092	364	8.70	39,102.69
PmbZIP86	RLN12841.1	PM09G12140	9	21889497–21890045	549	549	183	9.67	20,244.55
PmbZIP87	RLN13025.1	PM09G17690	9	43522067–43524827	2761	423	141	10.16	15,149.82
PmbZIP88	RLN12905.1	PM09G18590	9	44792601–44796981	4381	1257	419	9.11	44,598.29
PmbZIP89	RLN12453.1	PM09G18780	9	45041072–45044603	3532	912	304	4.83	32,658.00
PmbZIP90	RLN13126.1	PM09G20800	9	47002029–47003859	1831	849	283	6.24	30,013.48
PmbZIP91	RLN12623.1	PM09G24100	9	50246233–50248502	2270	477	159	9.00	17,365.70
PmbZIP92	RLM55632.1	PM10G06050	10	4576359–4581176	4818	1113	371	9.00	40,165.13
PmbZIP93	RLM54848.1	PM10G06130	10	4623806–4625917	2112	1524	508	9.16	55,261.12
PmbZIP94	RLM55196.1	PM10G10750	10	10699576–10701422	1847	735	245	10.88	27,271.78
PmbZIP95	RLM54794.1	PM10G15500	10	26027305–26029674	2370	525	175	9.75	18,810.58
PmbZIP96	RLM56218.1	PM10G16250	10	26722529–26726897	4369	1362	454	9.13	48,594.99
PmbZIP97	RLM55027.1	PM10G16410	10	26862899–26864549	1651	897	299	5.43	32,538.26
PmbZIP98	RLM55428.1	PM10G18250	10	28349450–28351117	1668	867	289	6.07	30,673.21
PmbZIP99	RLM56082.1	PM10G21570	10	31094548–31096463	1916	483	161	8.62	17,579.92
PmbZIP100	RLN09938.1	PM11G00290	11	452913–453281	369	369	123	8.97	13,810.34
PmbZIP101	RLN08312.1	PM11G02090	11	2470988–2476530	5543	1374	458	8.92	49,484.85
PmbZIP102	RLN07652.1	PM11G02220	11	2606668–2616433	9766	1035	345	4.52	35,333.37
PmbZIP103	RLN09078.1	PM11G02900	11	3245503–3248289	2787	939	313	5.42	33,095.76
PmbZIP104	RLN07125.1	PM11G13460	11	25503439–25506558	3120	1080	360	5.79	38,511.02
PmbZIP105	RLN09230.1	PM11G13790	11	25784422–25786801	2380	516	172	6.29	17,688.93
PmbZIP106	RLN07329.1	PM11G19430	11	38531228–38532874	1647	765	255	5.78	27,050.34
PmbZIP107	RLN08025.1	PM11G20550	11	40200637–40206785	6149	1797	599	9.76	65,299.21
PmbZIP108	RLN07962.1	PM11G20760	11	40430132–40431310	1179	567	189	9.02	20,553.29
PmbZIP109	RLN09062.1	PM11G24760	11	43626713–43632822	6110	1062	354	6.48	37,768.04
PmbZIP110	RLN07622.1	PM11G27250	11	46387650–46388183	534	534	178	8.65	20,643.10
PmbZIP111	RLM78368.1	PM12G01810	12	1318893–1319414	522	522	174	6.83	17,739.04
PmbZIP112	RLM78875.1	PM12G02550	12	1757193–1761266	4074	1362	454	8.20	47,603.58
PmbZIP113	RLM80422.1	PM12G04340	12	3257399–3260279	2881	924	308	5.20	32,615.15
PmbZIP114	RLM79681.1	PM12G05380	12	4052057–4052425	369	369	123	9.35	13,871.48
PmbZIP115	RLM80771.1	PM12G06150	12	4748641–4750662	2022	414	138	10.11	15,166.99
PmbZIP116	RLM79948.1	PM12G08110	12	6857609–6859787	2179	762	254	5.78	26,937.22
PmbZIP117	RLM79671.1	PM12G27010	12	38519464–38520329	866	510	170	7.93	19,646.96
PmbZIP118	RLM78209.1	PM12G28080	12	39453559–39458489	4931	1332	444	9.41	47,492.18
PmbZIP119	RLM79651.1	PM12G29310	12	40484976–40490089	5114	513	171	4.93	18,799.88
PmbZIP120	RLM80583.1	PM12G31970	12	42325452–42326207	756	519	173	9.00	18,729.10
PmbZIP121	RLN05398.1	PM13G04720	13	5033013–5042282	9270	1434	478	7.24	51,950.15
PmbZIP122	RLN05595.1	PM13G09960	13	25084063–25085042	980	261	87	9.65	9705.76
PmbZIP123	RLN04073.1	PM13G14010	13	35015891–35017421	1531	1209	403	6.37	42,408.97
PmbZIP124	RLN05004.1	PM13G14470	13	35292857–35295671	2815	810	270	9.25	28,271.05
PmbZIP125	RLN04831.1	PM13G19990	13	41033378–41036302	2925	999	333	5.76	34,879.32
PmbZIP126	RLN04579.1	PM13G20830	13	41770762–41771759	998	675	225	8.54	24,211.82
PmbZIP127	RLN03801.1	PM13G24510	13	45184706–45186681	1976	1218	406	6.61	43,025.01
PmbZIP128	RLN03549.1	PM13G25090	13	45582601–45583142	542	432	144	10.02	15,452.52
PmbZIP129	RLM61107.1	PM14G04080	14	3139207–3146477	7271	1263	421	6.53	45,595.62
PmbZIP130	RLM60470.1	PM14G09560	14	20687606–20688770	1165	441	147	9.34	16,666.93
PmbZIP131	RLM60601.1	PM14G16770	14	29385478–29388989	3512	1218	406	5.52	42,836.29
PmbZIP132	RLM61427.1	PM14G17480	14	30010114–30011044	931	654	218	8.82	23,453.05
PmbZIP133	RLM60401.1	PM14G19800	14	31888088–31890896	2809	1128	376	6.32	39,473.30
PmbZIP134	RLM61133.1	PM14G19990	14	32037397–32038999	1603	1227	409	6.43	43,304.39
PmbZIP135	RLM61596.1	PM14G21450	14	33079164–33079675	512	453	151	9.55	16,265.44
PmbZIP136	RLM74471.1	PM15G12970	15	28520231–28523482	3252	690	230	9.83	24,604.94
PmbZIP137	RLM74357.1	PM15G25820	15	39118732–39121503	2772	1608	536	7.97	58,416.77
PmbZIP138	RLM65300.1	PM16G12130	16	25119044–25121594	2551	786	262	5.10	27,207.24
PmbZIP139	RLM66084.1	PM16G21310	16	31781217–31790325	9109	1203	401	6.18	44,727.90
PmbZIP140	RLM70069.1	PM17G01920	17	1423232–1427747	4516	1389	463	6.25	50,766.87
PmbZIP141	RLM69114.1	PM17G02070	17	1594724–1595732	1009	675	225	8.98	24,596.61
PmbZIP142	RLM59076.1	PM18G00820	18	648118–653777	5660	1431	477	6.09	52,218.33
PmbZIP143	RLM57820.1	PM18G00930	18	761889–762898	1010	648	216	8.61	23,467.31
PmbZIP144	RLM58717.1	PM18G01220	18	1078921–1083128	4208	993	331	6.37	35,578.56

3.2. Phylogenetic and Sequence Conservation Analysis of PmbZIPs

A phylogenetic analysis was performed with all 144 identified PmbZIP proteins, as well as 75 Arabidopsis and 78 foxtail millet bZIP family members (Figure 1 and Figure S1), using the neighbor-joining (NJ) algorithm in MEGA software (MEGA6.06). The 144 PmbZIP genes were divided into ten groups (designated as A to E, G, H, I, S, and U) according to the subfamilies classified for Arabidopsis. Based on phylogenetic relationships, Group S contains 29 members and is the largest subfamily, and the smallest subfamilies are Groups B, E, and U, with only 3 members in each. It is interesting to note that no PmbZIP member was assigned to Group F. A total of 22 PmbZIPs were classified as belonging to Group A, 25 to Group D, 23 to Group I, 21 to Group G, 8 to Group C, and 7 to Group H.

Figure 1.

Phylogenetic analysis of bZIP transcription factors in Arabidopsis thaliana, Setaria italica, and P. miliaceum. A maximum likelihood phylogenetic tree was constructed using the full-length bZIP protein sequences from A. thaliana (At), S. italica (Si), and P. miliaceum (Pm). The bZIP proteins were classified into 11 subgroups, each represented by a distinct color. Subgroup names are indicated outside the tree. The grouping was supported by sequence similarity and domain conservation, revealing evolutionary relationships and potential functional conservation among bZIP family members across the three species.

3.3. Motif and Structural Analysis of PmbZIPs

To investigate the protein sequence features of PmbZIPs, 12 different motifs were identified in PmbZIPs (Figure 2), with lengths ranging from 21 to 50 aa. The phylogenetic analysis showed that the same clusters of PmbZIPs had similar conserved domain compositions, and obvious differences between different groups were also found. Motif 1, as a “basic region” of the bZIP domain, existed in all PmbZIPs. Additionally, motifs 4 and 7, as two different “leucine zippers” of the bZIP domain, existed in Groups B, C, E, G, I, and S and Groups A, D, H, and U, respectively. Specifically, the PmbZIPs in Group D contain the most motif types, including motifs 1, 2, 3, 5, 7, 8, and 11. However, only motif 1 was present in PmbZIP135, PmbZIP128, PmbZIP38, and PmbZIP23 in Group S. This may be the reason why different subfamilies have different functions.

Figure 2.

Motif analysis of P. miliaceum bZIP proteins. Twelve conserved motifs were identified. Each motif is represented by a colored box with a unique color corresponding to the motif number, as shown in the legend.

The structural diversity of the PmbZIP family was analyzed in terms of the exon/intron arrangement of the coding sequences. The number of introns in PmbZIPs ranged from zero to twelve. The detailed gene structure of PmbZIPs is pictured in Figure 3. Twelve introns were identified in PmbZIP104 and PmbZIP112, whereas 24 PmbZIPs were identified as intronless. Subgroups G and D generally contain more than seven introns, while subgroup S often has no introns. Most closely related PmbZIPs in the same class or subfamily share a similar gene structure in terms of the number of introns.

Figure 3.

Gene structural analysis of PmbZIPs. The exon–intron structures of bZIP genes were visualized to compare their gene organization. Green rounded rectangles represent exons, and black solid lines indicate introns.

3.4. Promoter Analysis of PmbZIPs

To predict the biological function of PmbZIPs, 2000 bp upstream sequences from the translation start sites of PmbZIPs were analyzed using the PlantCARE database (Figure 4). The promoter of each PmbZIP consists of several cis-acting elements, such as phytohormone-responsive elements, MYB binding sites, light-responsive elements, anoxic-specific inducibility elements, abiotic stress-responsive elements, defense- and stress-responsive elements, seed-specific regulation elements, and root-specific elements. As illustrated in Table 2, a light-responsive element was identified in the promoters of all PmbZIPs. An abscisic acid-responsive element, methyl jasmonate (MeJA)-responsive element, and meristem expression element were identified in the promoters of 135, 128, and 99 PmbZIP genes, respectively. The promoters of 66 PmbZIPs contained an auxin-responsive element, 75 PmbZIPs contained a gibberellin-responsive element, and 55 PmbZIPs contained a salicylic acid-responsive element. Additionally, an MYB binding site, defense- and stress-responsive elements, a low-temperature-responsive element, and a zein metabolism regulation element were all found in 97, 33, 64, and 46 PmbZIPs, respectively. In total, PmbZIP143 promoters contained 73 (maximum) cis-elements, which included 34 light-responsive elements, 17 abscisic acid-responsive elements, 14 MeJA-responsive elements, 7 anoxic-specific inducibility elements, and 1 meristem expression element. However, PmbZIP21 promoters only contained 11 cis-elements. These findings demonstrate that PmbZIPs might be associated with various transcriptional regulations involving development, hormones, and stress responses.

Figure 4.

Cis-element analysis of promoter regions of PmbZIPs. The cis-acting regulatory elements within the 2000 bp upstream promoter regions of bZIP genes were analyzed. Different cis-elements are represented by colored boxes, each corresponding to a specific functional category.

Table 2.

A summary of cis-elements in the PmbZIPs promoter.

Cis-Acting Element	Element Number	Gene Number
Abscisic acid responsiveness element	618	135
Anoxic specific inducibility element	220	90
Auxin-responsive element	95	66
Defense and stress responsiveness element	39	33
Endosperm expression element	29	25
Gibberellin-responsive element	108	75
Light responsive element	1655	144
Low-temperature responsiveness element	95	64
MeJA-responsiveness element	752	128
Meristem expression element	154	99
MYB binding site	175	97
Protein binding site	4	4
Rcircadian control element	40	30
Root specific element	7	5
Salicylic acid responsiveness element	72	50
Seed-specific regulation element	30	26
Wound-responsive element	8	8
Zein metabolism regulation element	61	46

3.5. Expression Analysis of PmbZIPs in Seed Germination Under Salt Stress

To explore the expression patterns of these millet bZIP genes, we used RNA-seq data of yumi1 (Y1) and yumi9 (Y9) under salt stress in the seed germination stage. Based on the millet RNA-seq data, 67 bZIP genes were detected in all three sampling stages at the gene level (Figure 5). This suggests that nearly half of bZIP genes are broadly expressed during millet germination and development. In addition, the fact that 18 bZIP genes have different expression levels suggests that these genes were induced by salt and have a vital function in tolerance responses. The heatmap analysis found that PmbZIP6, PmbZIP11, PmbZIP15, PmbZIP71, PmbZIP78, PmbZIP89, and PmbZIP97 are downregulated salinity-responsive genes (SRGs). Upregulated SRGs include PmbZIP26, PmbZIP30, PmbZIP33, PmbZIP65, PmbZIP70, PmbZIP104, PmbZIP107, PmbZIP113, PmbZIP118, PmbZIP125, and PmbZIP131. Interestingly, the differential expression of most SRGs occurs when water is imbibed for 3 h or during radicle protrusion (RAP) in the seed envelope stage, thus indicating that the seeds reinitiate metabolic processes and stress response in this period.

Figure 5.

Expression heatmap of 67 differentially expressed bZIP genes. Samples were collected from two P. miliaceum varieties, Y1 and Y9, under control (CK) and salt treatment (NA) conditions at three time points. Gene expression levels were normalized (log₂ (FPKM + 1)) and visualized to reveal temporal and treatment-specific expression patterns.

4. Discussion

In the immediate future, broomcorn millet will become a crucial crop that can help in diversifying agriculture and promoting a healthier diet for humans. Several plant bZIP transcription factor families have been identified and characterized extensively, which play a vital role in the regulation of growth, development, and environmental response. However, these gene families have not been reported in broomcorn millet. Therefore, searching for and identifying advantageous bZIP genes in millet will facilitate further basic scientific research on molecular breeding processes.

4.1. Characterization of Broomcorn Millet bZIP Gene Family

The genes involved in genome replication events can evolve into genes with new functions, which play an important role in expanding genome content and diversifying gene functions [62]. Previous research showed that the emergence of the broomcorn millet genome was the result of the ~5.6 MYA hybridization of two closely related genomes. Most Panicum species are polyploids native to the tropical/semi-arid regions of the world. Many gene families in the millet genome are double copies, most of which are retained by the parental species and single-copy genes of the parental species [6]. Since there are 78 bZIP genes in the foxtail millet genome, 144 bZIP genes were predicted to be in the P. miliaceum genome. In addition, many subfamily bZIP genes are double copies in broomcorn millet, except those in Groups B, E, F, and U; this is consistent with previous research. After genome duplication, nonfunctionalization (duplicated genes are silenced), subfunctionalization (function is partitioned between the new paralogs), and neofunctionalization (duplicated genes gain new functions) generally take place [63,64,65]. In this study, we found that Group F genes could have been lost or had changed functions during their evolution, thus suggesting that there was extensive gene loss during genome duplication.

A phylogenetic analysis of the PmbZIP gene family revealed that subfamilies A, D, and S contain a relatively large number of members that form well-supported and tightly clustered groups, thus suggesting potential functional conservation and lineage-specific expansion. Notably, the S subfamily appears to be divided into four smaller clades, which are interspersed among the members of the G, A, and C subfamilies. This branching pattern suggests that during evolution, some ancestral S-type bZIP members may have undergone functional divergence and structural differentiation, thus giving rise to new regulatory subgroups such as G, A, and C. These findings support the hypothesis that bZIP subfamily diversification has been driven by both sequence divergence and the acquisition of specialized regulatory roles in different stress and developmental contexts.

Among these subfamilies, Group A bZIP genes stand out due to their well-established roles in abscisic acid (ABA)-mediated stress responses. In our study, PmbZIP30 (PmABI5) and PmbZIP131, both belonging to Group A, were significantly induced by ABA during seed development in broomcorn millet [60]. This is consistent with findings in Arabidopsis and rice, where homologous Group A bZIPs (e.g., ABI5) act as core components in the ABA signaling pathway. Furthermore, cross-species evidence supports the involvement of Group A bZIPs in the conserved PP2C–SnRK2–AREB signaling module, which regulates plant responses to drought and other abiotic stresses. The promoter architecture and expression dynamics of Group A PmbZIP genes in this study reinforce the notion that their functional roles in ABA-dependent stress signaling are evolutionarily conserved. These results not only provide insight into the diversification and conservation of bZIP transcription factors in broomcorn millet but also highlight the adaptive significance of Group A members in stress tolerance.

4.2. Structural Analysis of PmbZIPs

The detailed understanding of the functional domain of A. thaliana and S. italica bZIPs enabled us to analyze similar domains within the broomcorn millet bZIP gene family. In this research, all 144 PmbZIP proteins contain the necessary basic domain, which provides the structural basis for their conserved function. Moreover, different subgroups have different ZIP motifs, which makes their functions differ. Groups A, D, H, and U have type I ZIP domains (motif 7, K-L-X7-R), and the main function of these subfamily genes is the regulation of biological and abiotic stresses. However, Groups B, C, E, G, I, and S have type II ZIP domains (motif 4, V-L-X8-R) that are involved in carbohydrate biosynthesis, post-transcriptional inhibition, development, and hormone synthesis. It has been reported that intron retention regulates protein isoform production, RNA stability and translation efficiency, and the rapid induction of expression via the post-transcriptional splicing of retained introns [66]. An analysis of the bZIP gene structure revealed that most intronless PmbZIPs occurred in Groups S and C, and a similar observation was reported for banana (Musa spp. L.) and switchgrass (P. virgatum L.) [67,68]. The PmbZIPs of subgroups A, C, and I, with relatively fewer introns, were associated with stress responses. This conclusion aligns with the results of the present and past studies [60].

4.3. Cis-Element Analysis in the Promoters of PmbZIPs

The cis-elements of different transcription factors have different functions. The number and form of cis-elements in promoter regions could play an essential role in the regulation of gene expression. The results illustrate that abiotic stress-related cis-elements, including abscisic acid-responsive elements, anoxic-specific inducibility elements, low-temperature-responsive elements, MeJA-responsive elements, MYB binding sites, salicylic acid-responsive elements, and wound-responsive elements, are major regulatory elements in the PmbZIP promoters activated by ABA, NaCl, or other forms of abiotic stress. In addition, many development-related cis-elements were also found, such as auxin-responsive elements, endosperm expression elements, gibberellin-responsive elements, root-specific elements, seed-specific regulation elements, and meristem expression elements. These findings suggest that light-responsive elements, abscisic acid-responsive elements, and MeJA-responsive elements play a vital role in transcriptional regulation in broomcorn millet. PmbZIP promoters present a lot of stress-responsive cis-elements and hormone response cis-elements, which indicates their potential roles in the response to stress and pathogen infections. Consequently, PmbZIPs are often taken as candidate genes to understand the responses to biotic stresses and plant development.

4.4. PmbZIP Involvement in Development and Stress Response

Previous reports have revealed that bZIP TFs function in many stress responses and development by regulating diverse biochemical and physiological pathways [17,69,70,71]. bZIP transcription factors possess different characteristics in different species. The overexpression of TabZIP15 in wheat can enhance root length and fresh weight during salt stress, thus suggesting that the TabZIP15 gene is involved in the regulation of wheat salt stress tolerance [72]. The homozygous T-DNA insertional mutants Osabf1-1 and Osabf1-2 are more sensitive in response to drought and salinity treatments than wild-type plants, and the OsNAC, OsLEA3, and OsABA45 genes are significantly suppressed in Osabf1 mutants. Hence, OsABF1 likely plays a positive role as an ABA-responsive transcription factor in abiotic stress signaling [26]. We incorporated and highlighted experimental data from our previous work, wherein PmbZIP30 was overexpressed in rice. The transgenic rice lines exhibited significantly shorter seed germination times compared to the wild-type, thus indicating a positive regulatory role of PmbZIP30 in seed germination and stress response. This functional validation supports the biological relevance of our bioinformatic findings (Figure S2). The overexpression of StbZIP65 in Arabidopsis enhanced salt tolerance [73]. However, some research shows that GmbZIP19 expression is significantly induced by ABA (abscisic acid), JA (jasmonic acid), and SA (salicylic acid) but is reduced under salt and drought stress conditions, thus suggesting that GmbZIP19 is a positive regulator of pathogen resistance and a negative regulator of salt and drought stress tolerance [74]. TabZIP6 can bind to the promoters of CBFs and decrease the expression of downstream COR genes in TabZIP6-overexpressing Arabidopsis seedlings; therefore, TabZIP6 is a negative regulator in the cold stress response [75].

In this study, a total of 144 PmbZIP genes were identified in the P. miliaceum genome and classified into eleven subfamilies based on phylogenetic relationships. Conserved motif and domain structure analyses revealed that the members within each subfamily exhibit high sequence conservation. The promoter analysis indicated that PmbZIP genes may be involved in multiple hormone signaling pathways and environmental stress responses, as reflected by the presence of diverse cis-acting regulatory elements, including MYB binding sites. The transcriptome analysis further showed that 18 PmbZIP genes were differentially expressed during seed germination under salt stress, thus suggesting their potential regulatory roles in abiotic stress adaptation. These findings provide a valuable resource for understanding the functional roles of bZIP transcription factors in broomcorn millet and lay the foundation for future stress resilience breeding and gene function studies.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/genes16070734/s1. Figure S1: Sequence conservation analysis of PmbZIPs. Figure S2: Overexpression of PmbZIP30 in rice accelerates seed germination.

Author Contributions

Writing—original draft preparation, P.A.; writing—review and editing, P.R., S.D. and P.A.; supervision, T.L. and Z.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Doctoral Research Fund of Longdong University, grant number XYBYZK2206.