Genome-wide analysis of the LRR-RLK genes in yerba mate (Ilex paraguariensis)

Abstract

Yerba mate is a significant crop for culture and industries across southern South America. However, increasing environmental challenges currently threaten its productivity, ecological stability, and socioeconomic role. We, therefore, focused on broadly characterizing the LRR-RLK genes in yerba mate predicting their potential roles in development, growth, and stress response, providing a molecular basis for crop management. We identified 226 LRR-RLK genes (94.7% on 20 chromosomes), diverse in structural features, but displaying the typical protein domain arrangement (SP|LRR|TM|K). Phylogenetic analysis classified these genes into 25 distinct homology groups, consistent with Arabidopsis. Subsequent functional and regulatory analyses included gene ontology annotation, subcellular localization, promoter regions survey, and expression profiling. Most LRR-RLK genes were annotated to key processes such as transferase activity, signaling, development, reproduction, and defense response, and predicted primarily to the plasma membrane. Promoters mostly harbored binding sites for ten transcription factor families associated with diverse stresses, hormones, growth, and development. Nearly 59% of LRR-RLK genes exhibited expression levels suggesting a basal regulatory role. Furthermore, 96.1% of LRR-RLK genes were differentially expressed under curly leaf disease, drought stress in leaves and roots, and leaves and roots comparison. These findings provide a comprehensive foundation for selecting candidate genes involved in stress adaptation.

Key words:
crop tree; environmental challenges; kinases; stress genes

Resumen

La yerba mate es un cultivo importante del sur Sudamericano, cuya productividad, ecología y rol socioeconómico están amenazados por crecientes desafíos ambientales. Caracterizamos los genes LRR-RLK en yerba mate basado en sus potenciales roles en el desarrollo, crecimiento y respuesta al estrés, proporcionando una base molecular para el manejo del cultivo. Identificamos 226 genes LRR-RLK (94,7% en 20 cromosomas), diversos estructuralmente, pero con disposición de dominios proteicos típica (SP|LRR|TM|K). El análisis filogenético los clasificó en 25 grupos de homología, consistente con Arabidopsis. Los análisis posteriores incluyeron anotación GO, localización subcelular, sondeo de promotores y perfiles de expresión. La mayoría de los genes LRR-RLK se anotaron a procesos clave como actividad de transferasa, señalización, desarrollo, reproducción y defensa, y se predijeron primariamente en la membrana plasmática. Los promotores albergaron principalmente sitios de unión para diez familias de factores de transcripción asociadas a estrés, hormonas, crecimiento y desarrollo. Cerca del 59% de los genes mostraron niveles de expresión que sugieren un rol regulatorio basal. Además, el 96,1% se expresó diferencialmente bajo condiciones de enfermedad del rulo, estrés por sequía en hojas y raíces, y comparación de hojas y raíces. Estos hallazgos sientan una base integral para la selección de genes candidatos involucrados en la adaptación al estrés.

Palabras clave:
árbol cultivado; desafíos ambientales; kinasas; genes de estrés

Introduction

The erva/yerba mate tree Ilex paraguariensis A. St.-Hil. (Aquifoliaceae) is a significant crop for industries in Argentina, Brazil, Paraguay, and Uruguay, where its dried leaves and twigs are widely consumed in traditional hot infusions (“mate” or “chimarrão”) and a cold version (“tereré”) (Pereira Croge et al. 2021). Beyond its cultural significance, the plant is rich in diverse bioactive compounds, including terpenes, flavonoids, phenolics, and methylxanthines (such as caffeine), which contribute to its beneficial effects and are expected to expand its technological applications (Gullón et al. 2018; Gerber et al. 2023). While I. paraguariensis is native and adapted to the Atlantic Forest biome, its widespread cultivation in full-sun monoculture exposes it to considerable environmental challenges (Montagnini et al. 2011). In this context, yerba mate plantations are negatively affected by diverse biotic stressors, including insect and acari pests, as well as fungal and viral pathogens, besides abiotic stresses such as intense direct solar radiation and drought that significantly reduce productivity (Sosa et al. 2011; Debat et al. 2014; Rybak et al. 2014; Bejerman et al. 2017, 2020; Bergottini et al. 2017; Gortari et al. 2020).

Plants, as sessile organisms, rely on a sophisticated molecular machinery to perceive and respond to these challenging environmental conditions, while also facilitating normal development and growth (Vierling 1991; Rajan & D’Silva 2009). Among these crucial components, protein kinases play a central role in signal transduction networks, and the leucine-rich repeat receptor-like kinase (LRR-RLK) proteins constitute a significant part of this machinery (Man et al. 2020). The LRR-RLK gene clade represents the largest subfamily within the Receptor-Like Kinase/Pelle (RLK-Pelle) protein family in higher plants, a monophyletic group that can comprise hundreds to over a thousand members per species (Shiu & Bleecker 2001; Gish & Clark 2011; Lehti-Shiu & Shiu 2012; Yin et al. 2024). For instance, the RLK-Pelle family alone accounts for over 600 members in Arabidopsis thaliana (L.) Heynh. and more than 1,100 in Oryza sativa L. (rice) (Liu et al. 2024). LRR-RLK proteins are characterized by a tripartite structure: an extracellular LRR domain responsible for ligand binding, a single-pass transmembrane region (TM), and an intracellular protein kinase catalytic domain (K) (Bezerra-Neto et al. 2020). The diversity in the number and arrangement of LRR motifs enables these receptors to perceive a wide array of extracellular signals (Li et al. 2018). LRR-RLKs are typically classified into 15 groups (I to XV) based on the phylogenetic relationships of their kinase domain and the arrangement of LRR motifs (Shiu & Bleecker 2001; Lehti-Shiu & Shiu 2012). Functionally, LRR-RLK proteins are pivotal in various aspects of plant biology, including regulating growth, morphogenesis, organogenesis, and hormone signaling, as well as mediating robust defense responses to biotic and abiotic stresses, often acting as pattern recognition receptors (PRRs) and constituting disease resistance (R) gene candidates (Tang et al. 2010; Gish & Clark 2011; Bezerra-Neto et al. 2020; Yan et al. 2023).

Recent advances in genomics have provided unprecedented insights into the genetic basis of traits in economically important plant species. The nuclear genome of I. paraguariensis has recently been assembled to chromosome-level, revealing a total length of 872.9 Mb and containing 41.9 thousand protein-coding genes (Aguilera & Grabiele 2025). Comparative genomic analyses have shown a lineage-specific whole-genome duplication event during the early evolutionary history of Ilex in addition to the ancient hexaploidization event shared by core eudicots (Xu et al. 2022; Yao et al. 2022). These whole-genome duplication events are recognized as key evolutionary mechanisms that provide the raw genetic material for gene family expansion and contribute to the evolution of metabolic and developmental complexity (Lynch & Conery 2000; Shiu & Bleecker 2003). Given the significant expansion of kinase gene families in plants, particularly the RLK-Pelle family (Shiu & Bleecker 2003), these genomic features strongly suggest that I. paraguariensis possesses an expanded and diversified repertoire of LRR-RLK genes, which are likely crucial for its resilience and adaptive success in challenging environments.

For the accurate management of the yerba mate crop, it is essential to understand the genetic basis of stress responses in this species, considering the generally stressful environmental conditions of its plantations (Aguilera et al. 2023). To support the achievement of this goal, we comprehensively characterized the LRR-RLK genes in yerba mate and predicted their possible roles. For this purpose, we carried out a genome-wide identification and mapping at the chromosome-level assembly YMM1 of yerba mate (Aguilera & Grabiele 2025), characterized the structural-functional features and cis-acting regulatory regions of genes, proposed a phylogenetically based classification, and performed expression analysis of the LRR-RLK subfamily of RLKs in this important crop tree species.

Material and Methods

Identification, classification and structural-functional characterization of yerba mate LRR-RLK genes

Downstream analyses were conducted using our annotated nuclear chromosome-level assembly YMM1 of yerba mate (Aguilera & Grabiele 2025), deposited at the NGDC (<https://ngdc.cncb.ac.cn/>) under accession OMIX007912 of BioProject PRJCA032295.

Kinase superfamily genes were identified by significant protein sequence hits to the kinase domains PF00069 and PF07714 in the PFAM database (<https://pfam.xfam.org/>). These were classified into well-supported gene families by comparing their protein sequences to a set of Hidden Markov Models (HMMs) developed by Lehti-Shiu & Shiu (2012) and integrated into the iTAK program (<http://itak.feilab.net/cgi-bin/itak/index.cgi>; Zheng et al. 2016). The RLK-Pelle kinase family was further classified into subfamilies based on phylogenetic relationships of their kinase domains, including LRR-RLK subfamilies with similar domain organization.

To obtain a representative repertoire of yerba mate LRR-RLK genes, we performed homology-based searches via BLASTP at the standard cutoff 1e-05 (Altschul et al. 1990), using the whole proteome and characterized LRR-RLK genes of A. thaliana. The A. thaliana LRR-RLK proteins with their corresponding gene IDs and classification, were obtained from the iTAK database (228 genes) and RLKdb (<https://biotec.njau.edu.cn/rlkdb/>; Yin et al. 2024) (225 genes) and merged into a non-redundant set of 241 proteins (285 sequences including isoforms). Candidate LRR-RLK genes were also searched via LRR and kinase domain signatures within the eggNOG-mapper (<http://eggnog-mapper.embl.de/>; Cantalapiedra et al. 2021) orthology-based annotation data from Aguilera & Grabiele (2025).

To confirm the joint presence of LRR, TM, and kinase domains among other characteristic domains, a structural-functional annotation of the candidate LRR-RLK genes was performed via InterPro (Blum et al. 2024) in the Geneious environment (Dotmatics), considering PFAM, PHOBIUS, PRINTS, PROSITE, SIGNALP_EUK, SMART, Superfamily, and TMHMM member databases. Based on incomplete domain architecture, 52 candidate genes were identified for remodeling (Tab. S1). This vital step was conducted via FGENESH at the Dicot plants mode (Solovyev et al. 2006; <https://usegalaxy.org.au/>), and the resulting proteins were re-annotated via InterPro. An updated version of YMM1, including 32 remodeled LRR-RLK genes, was deposited at Mendeley Data (Aguilera & Grabiele 2026). Finally, only protein sequences containing LRR, TM, and kinase domains were considered LRR-RLK genes, including those lacking the LRR domain if they shared homology with Arabidopsis LRR-RLK genes with similar domain constitution. Gene models reported as incomplete after FGENESH, i.e., sole displaying partial or complete kinase domains, were considered as pseudogenes (Tab. S1), and were excluded from successive downstream analysis. Gene models displaying kinase and TM domains but lacking the LRR domain and holding a non-cytoplasmatic domain in the corresponding region were classified as possible pseudogenes (Tab. S1). BLASTP analyses (cutoff 1e-05) were conducted within yerba mate and at NCBI to confirm the status of pseudogenes and non-LRR-RLK genes versus LRR-RLK genes.

Gene ontology (GO) annotation included eggNOG-mapper for GO Full Term IDs, with subsequent mapping of GO Slim Term IDs and Labels via GoSlimmer (1.0.1; Faria 2017) to finally get ontology files (<http://geneontology.org/docs/download-ontology>) in the Galaxy environment (<https://usegalaxy.eu/>; The Galaxy Community 2024). Subcellular localization was determined via WoLF PSORT (0.0.12; Horton et al. 2007) in Galaxy.

To achieve a group classification, an unrooted maximum likelihood (ML) phylogenetic tree was built using complete amino acid sequences of yerba mate and A. thaliana LRR-RLK proteins. Multiple sequence alignment was performed via MUSCLE (3.8.31), and phylogenetic relationships were inferred via FastTree (2.1.5), using the GTR+G model and 1,000 resamples (support values: 0-lowest to 1-highest) within Geneious, where exon-intron structures were also visualized and analyzed.

Chromosomal mapping and cis-acting regulatory regions analysis of yerba mate LRR-RLK genes

LRR-RLK genes were mapped onto the chromosomes of the YMM1 assembly, and their distribution was visualized and further analyzed using Geneious. Promoter regions were submitted for transcription factor families binding site (TFFBS) prediction, considering 2,000 nucleotides upstream and downstream of start and stop codons, respectively (Chow et al. 2016). This was done using the 674 non-redundant, high-quality MEME binding motifs from PlantTFDB (5.0; Jin et al. 2017) and the FIMO search engine of MEME Suite (5.5.7; Bailey et al. 2015), with motifs considered valid at p-value ≤ 1e-4 and q-value ≤ 0.05 as usual.

Expression pattern analysis of yerba mate LRR-RLK genes

We utilized 26 paired-end RNA-seq libraries of yerba mate (127 Gb total), retrieved from NCBI (Tab. S2), comprising samples from leaves (16), roots (9), and a seedling (1). Reads were pre-processed using Trimmomatic (0.38.0; Bolger et al. 2014) in Galaxy. This step removed adapters and low-quality bases via sliding window trimming (window size: 4 bases; quality score: 20), retaining only the paired-end data for subsequent analysis. Transcript abundances were quantified across samples using Kallisto quant (0.48.0; Bray et al. 2016) in Galaxy, incorporating 100 bootstrap replicates and sequence-based bias correction. Abundances were normalized using Transcripts Per Million (TPM), calculated considering the entire set of yerba mate genes. Differential expression (DE) analysis was performed on the Kallisto count tables using DESeq2 (2.11.40.8; Love et al. 2014) in Galaxy. Changes in gene expression were considered statistically significant at the standard False Discovery Rate (FDR)-adjusted p-value threshold ≤ 0.05 (Storey & Tibshirani 2003). The magnitude of gene expression change was quantified using the logarithm to base 2 of the fold change (log₂ FC), which was classified into three categories based on magnitude: strong (|log₂ FC| ≥ 1; +1: 100% upregulated gene; -1: 50% downregulated gene), moderate (0.58 ≤ |log2 FC| < 1; +0.58: 50% upregulated gene; -0.58: 33% downregulated gene) and light change (|log2 FC| < 0.58).

Results

We identified 1,200 candidate kinase genes in yerba mate, classified into 20 gene families (Fig. 1a). The RLK-Pelle family was the largest, accounting for 66.7% of all kinase genes, with LRR-RLK subfamilies representing 30.6% of this group (Fig. 1a-b).

Figure 1
a-f. Characterization of Kinase and LRR-RLK genes in yerba mate - a. percentage distribution of genes within Kinases families; b. percentage distribution of genes within RLK-Pelle subfamilies; c. characteristic domain arrangement of LRR-RLK genes; d. chromosomal distribution of 226 LRR-RLK genes (green bars), 17 confirmed pseudogenes and three possible pseudogenes (blue dots) and 24 gene clusters (red dots). Green values at the bottom correspond to the number of LRR-RLK genes per chromosome; e. distribution of the 246 LRR-RLK genes sensu lato into functional categories; f. distribution graph of distinct structural features associated with the 226 LRR-RLK genes. #: number.

Yerba mate has 226 LRR-RLK genes, with 153 currently annotated alternative splice variants (Tabs. 1-2). Of these, 221 displayed the typical LRR, TM, and kinase domain arrangement (Fig. 1c; Tab. 2; Tab. S1; File S1). Five genes had TM and kinase domains but lacked the LRR domain, similar to their A. thaliana homologous. Four of these also had a non-cytoplasmatic domain in the corresponding LRR region, matching their respective homolog (Tab. S1; File S1). Most LRR-RLK genes (94.7%; 214) were distributed across 20 chromosomes (1-20), while 12 were located on artificial scaffold 21 (Tab. 1; Fig. 1d). Additionally, 17 LRR-RLK pseudogenes that lacked the LRR domain were identified -16 of which also lacked the TM domain- while retaining a complete or partial K domain (Fig. 1d-e; Tab. S1; File S2). Nine of these pseudogenes were highly comparable to active LRR-RLK genes of yerba mate, mostly in their vicinity, while seven resembled active or inactive LRR-RLK genes in other species. Finally, a retrotransposon-caused pseudogene was also identified (File S3).

Table 1
LRR-RLK genes of yerba mate: major features.

Ch	ID1	begin	end	D	status	gene length (nt)	CDS length (nt)	intron length (%)2	# coding exons	group	cluster3
1	Ipa_000073	546076	551985	R	gene	5910	2322	60.7	7	VI-1
1	Ipa_000218.2	2493424	2495781	R	gene	2358	1923	18.4	2	III
1	Ipa_000278	3247936	3249747	F	gene	1812	1812	0	1	Xa
1	Ipa_000350	4181247	4189667	F	gene	8421	2673	68.3	7	XI-1	1
1	Ipa_000351	4195312	4199502	F	gene	4191	2361	43.7	2	XI-1	1
1	Ipa_000353.2	4212278	4215245	F	gene	2968	2889	2.7	2	XI-1	1
1	Ipa_000355.2	4217716	4221065	F	gene	3350	3294	1.7	2	XI-1	1
1	Ipa_000472	5336086	5339496	F	gene	3411	3411	0	1	XV
1	Ipa_000539.2	7831736	7836218	F	gene	4483	1266	71.8	8	I-2
1	Ipa_000586	8651471	8654555	R	gene	3085	1506	51.2	10	II
1	Ipa_000861	13286537	13289987	R	gene	3451	1977	42.7	2	III	2
1	Ipa_000888.2	13802401	13807789	F	gene	5389	2520	53.2	4	III	2
1	Ipa_001164	18669461	18673358	R	gene	3898	2964	24.0	2	XI-1	3
1	Ipa_001166	18771852	18775860	R	gene	4009	3051	23.9	2	XI-1	3
1	Ipa_001868	40997372	41000891	F	gene	3520	3258	7.4	2	Xb-2
1	Ipa_001933.2	43238021	43241251	F	gene	3231	2778	14.0	3	XII-1
1	Ipa_002927	64682650	64688474	R	gene	5825	2226	61.8	11	VI-2
1	Ipa_002938	64979068	64981976	F	gene	2909	2655	8.7	2	XI-1
1	Ipa_002957	65177765	65180792	R	gene	3028	2631	13.1	2	III
1	Ipa_003020	65781042	65784579	R	gene	3538	2679	24.3	3	VII-1
1	Ipa_003080	66483607	66487116	F	gene	3510	3087	12.1	2	XI-1
1	Ipa_003102	66764596	66768528	F	gene	3933	2580	34.4	5	IV	4
1	Ipa_003131	67085484	67089416	F	gene	3933	2580	34.4	5	IV	4
1	Ipa_003146	67292667	67299605	R	gene	6939	2964	57.3	27	XIIIb
1	Ipa_003216	68049281	68053189	F	gene	3909	3909	0	1	Xb-1
1	Ipa_003354	69462597	69465573	F	gene	2977	2070	30.5	4	III
2	Ipa_003646.2	702143	705517	R	gene	3375	3117	7.6	2	XII-1
2	Ipa_003922	3639970	3644227	F	gene	4258	2112	50.4	5	IV
2	Ipa_004205	6715411	6731307	F	gene	15897	4413	72.2	14	III
2	Ipa_004435	10133727	10137226	R	gene	3500	3081	12.0	2	XI-1	5
2	Ipa_004450	10337501	10341885	F	gene	4385	2889	34.1	2	XI-1	5
2	Ipa_004982.2	22117970	22124793	R	gene	6824	2172	68.2	21	XIIIb
2	Ipa_005081	29321559	29331378	R	gene	9820	3456	64.8	5	XI-1
2	Ipa_005095	31263107	31291709	R	pseudo	28603	612	97.9	3
2	Ipa_005097	31566374	31583683	R	gene	17310	1911	89.0	4	III
2	Ipa_005238	39967814	40000564	R	gene	32751	3135	90.4	24	IX
2	Ipa_005765	54905595	54909536	F	gene	3942	3480	11.7	2	XII-1
2	Ipa_005844	56279459	56320744	F	gene	41286	3072	92.6	28	IX	6
2	Ipa_005859	56683833	56700137	R	gene	16305	3045	81.3	25	IX	6
2	Ipa_005860	56718640	56731135	R	gene	12496	3075	75.4	24	IX	6
2	Ipa_005861	56754077	56775900	R	gene	21824	2973	86.4	25	IX	6
2	Ipa_005862	56808103	56833211	R	gene	25109	3321	86.8	24	IX	6
2	Ipa_005912	57439137	57442736	R	gene	3600	2034	43.5	2	VII-2	7
2	Ipa_005916	57525971	57533254	R	gene	7284	2163	70.3	13	VI-2	7
2	Ipa_006031	59382313	59384907	R	gene	2595	2595	0	1	XI-1
2	Ipa_006326	63429186	63441610	F	gene	12425	2013	83.8	17	V
3	Ipa_006505	1262626	1270155	R	gene	7530	1899	74.8	14	V
3	Ipa_006712	3618392	3629128	R	gene	10737	2364	78.0	19	V
3	Ipa_006861	5309566	5317192	F	gene	7627	2490	67.4	16	VI-2
3	Ipa_006873	5504086	5513166	F	gene	9081	1968	78.3	15	II
3	Ipa_007141	10249800	10254252	F	gene	4453	2991	32.8	4	III
3	Ipa_007476	26773013	26777176	F	gene	4164	1914	54.0	3	III
3	Ipa_007761	38375763	38378342	R	gene	2580	2580	0	1	VII-2
3	Ipa_007814	39476147	39479796	R	gene	3650	2892	20.8	2	XI-1
3	Ipa_008083	44143789	44147169	R	gene	3381	1923	43.1	2	III
3	Ipa_008398.2	48534352	48537486	F	gene	3135	3054	2.6	2	XI-1
3	Ipa_008508	50032068	50035781	F	gene	3714	1965	47.1	2	III
3	Ipa_008794	53582011	53586381	F	gene	4371	2619	40.1	5	VII-2
3	Ipa_009231	59489886	59493994	R	gene	4109	3060	25.5	4	III
4	Ipa_009688	3846889	3850971	F	gene	4083	3681	9.8	2	XI-1	8
4	Ipa_009708	4057043	4060643	R	gene	3601	3384	6.0	2	XI-1	8
4	Ipa_009758	4579512	4585294	F	gene	5783	2721	52.9	16	I-1	9
4	Ipa_009760	4590926	4600857	F	gene	9932	2997	69.8	23	I-1	9
4	Ipa_009894	6138624	6142030	R	gene	3407	2031	40.4	5	IX
4	Ipa_009918	6332208	6333185	F	pseudo	978	978	0	1
4	Ipa_009967	6826626	6837136	F	gene	10511	2127	79.8	5	III
4	Ipa_010052	7850650	7855886	F	gene	5237	2124	59.4	9	VI-2
4	Ipa_010096.2	8267228	8270102	F	gene	2875	1950	32.2	5	XII-1
4	Ipa_010679	17078901	17085094	R	gene	6194	3084	50.2	3	III
4	Ipa_011133	35080249	35083365	F	gene	3117	3117	0	1	Xb-1
4	Ipa_011255	37040486	37042181	F	gene	1696	915	46.0	5	XIIIa
4	Ipa_011321	38176181	38180365	F	gene	4185	3147	24.8	3	XI-1
4	Ipa_011702	44361198	44376785	F	gene	15588	3462	77.8	25	IX
4	Ipa_011975.2	47204471	47207847	F	gene	3377	3276	3.0	2	XI-1
5	Ipa_013012	15230408	15233145	R	gene	2738	2556	6.6	3	VII-1
5	Ipa_013475	33754433	33775647	F	gene	21215	2556	88.0	21	VIII-1	10
5	Ipa_013476	33777593	33796160	F	gene	18568	2532	86.4	15	VIII-1	10
5	Ipa_013477	33889500	33917328	F	gene	27829	3660	86.8	31	VIII-1	10
5	Ipa_013479	33934501	33990093	F	gene	55593	2940	94.7	21	VIII-1	10
5	Ipa_013481	34031850	34044425	F	gene	12576	2784	77.9	18	VIII-1	10
5	Ipa_013483	34045853	34047176	F	pseudo	1324	864	34.7	3		10
5	Ipa_013487	34109185	34142486	F	gene	33302	3066	90.8	20	VIII-1	10
5	Ipa_013621	36773039	36778108	F	gene	5070	2988	41.1	2	XI-1
5	Ipa_013766	38841616	38846235	F	gene	4620	1107	76.0	8	II
5	Ipa_013789	39142520	39144944	F	gene	2425	1911	21.2	2	III
5	Ipa_014033	42262802	42272241	F	gene	9440	2361	75.0	6	III
5	Ipa_014122	43129657	43132989	F	pseudoP	3333	1413	57.6	3
5	Ipa_014220	44074454	44081696	R	gene	7243	2544	64.9	6	XIV
6	Ipa_014689	2843745	2852194	F	gene	8450	2709	67.9	4	XIV
6	Ipa_015017.2	7216608	7220045	R	gene	3438	2949	14.2	2	XI-1
6	Ipa_015189	10710416	10730245	R	gene	19830	1404	92.9	10	II
6	Ipa_015785	26219226	26227353	R	pseudo	8128	1770	78.2	5
6	Ipa_016107.2	32381719	32386078	R	gene	4360	3060	29.8	4	XI-1
6	Ipa_016500	37259332	37262768	F	gene	3437	2049	40.4	4	IV
7	Ipa_017440	8242662	8246370	R	gene	3709	3282	11.5	3	XI-1
7	Ipa_017534	10629718	10634053	F	gene	4336	2766	36.2	2	XI-1
7	Ipa_017591	12218832	12221775	R	gene	2944	1785	39.4	3	IX
7	Ipa_018164	32043880	32047269	F	gene	3390	3390	0	1	XV
7	Ipa_018500	36913266	36916870	R	pseudo	3605	966	73.2	2
7	Ipa_018563	37742022	37745882	R	gene	3861	2877	25.5	2	IX
8	Ipa_018615	306181	309372	F	gene	3192	3192	0	1	XV
8	Ipa_018858	2568005	2571710	F	gene	3706	3012	18.7	2	XI-1	11
8	Ipa_018860	2583822	2590812	F	gene	6991	2694	61.5	5	XI-1	11
8	Ipa_018861.2	2595016	2596247	F	pseudoP	1232	1092	11.4	2		11
8	Ipa_018955	3341700	3345116	F	gene	3417	3417	0	1	XV
8	Ipa_019086	4656684	4659249	R	gene	2566	1932	24.7	2	III
8	Ipa_019192.2	5561985	5565643	R	gene	3659	3048	16.7	2	XI-1
8	Ipa_019222	5923067	5933017	F	gene	9951	2865	71.2	16	I-1
8	Ipa_019510	9314902	9318315	F	gene	3414	3414	0	1	VII-3
8	Ipa_019677	11344267	11348791	R	gene	4525	1986	56.1	13	VI-2
8	Ipa_019881	14547314	14551208	F	gene	3895	2943	24.4	3	IX
8	Ipa_019906	15544434	15551248	F	gene	6815	1959	71.3	2	III
8	Ipa_020099	20167042	20190075	F	gene	23034	1455	93.7	11	II
8	Ipa_020248	25322174	25329954	R	gene	7781	3204	58.8	3	III
8	Ipa_020335	32336193	32339795	F	gene	3603	3603	0	1	Xb-1
9	Ipa_020532	359678	362770	R	gene	3093	2001	35.3	5	III
9	Ipa_021531	21794755	21798341	R	pseudo	3587	543	84.9	3
9	Ipa_021553	22076362	22080205	F	gene	3844	3702	3.7	2	Xb-1
9	Ipa_021836	26092986	26098434	F	gene	5449	2184	59.9	17	V
10	Ipa_022154	425683	442479	F	gene	16797	3108	81.5	24	IX
10	Ipa_022204	854927	858616	F	gene	3690	3690	0	1	Xb-1
10	Ipa_022270.2	1539006	1542382	R	gene	3377	3285	2.7	2	XI-1
10	Ipa_022381	2811763	2814109	F	gene	2347	1812	22.8	2	III
10	Ipa_022765	6597150	6616159	F	gene	19010	2922	84.6	23	IX
10	Ipa_023263	12834256	12850067	R	gene	15812	2349	85.1	19	V
10	Ipa_023492	17579864	17583541	R	gene	3678	2952	19.7	4	XI-1
10	Ipa_023675	27999982	28004141	F	gene	4160	3033	27.1	4	XI-1
11	Ipa_023979.2	9871376	9876612	R	gene	5237	4629	11.6	5	XII-1
11	Ipa_023998	10678684	10682565	R	gene	3882	3882	0	1	Xb-1
11	Ipa_024030	11605350	11609397	F	gene	4048	3030	25.1	2	VII-1
11	Ipa_024182	15152506	15158426	F	gene	5921	2760	53.4	4	IX
11	Ipa_024399	20538520	20542125	F	gene	3606	3510	2.7	2	XI-1	12
11	Ipa_024409	20633957	20640247	R	gene	6291	3327	47.1	2	XI-1	12
11	Ipa_025087	29699499	29703451	R	gene	3953	3264	17.4	2	XI-1
12	Ipa_025180	2706652	2727875	R	gene	21224	2727	87.2	20	VIII-1
12	Ipa_025183	2817051	2820122	R	gene	3072	3072	0	1	Xb-1
12	Ipa_025467	18393814	18397740	R	gene	3927	2172	44.7	2	III
12	Ipa_025523	19219125	19226909	F	gene	7785	2688	65.5	16	I-1	13
12	Ipa_025537	19430642	19438592	F	gene	7951	2589	67.4	16	I-1	13
12	Ipa_025666	21108341	21111965	R	gene	3625	1194	67.1	10	II
12	Ipa_025785	23106143	23107963	F	gene	1821	1821	0	1	Xa
12	Ipa_025837	23736424	23739764	R	gene	3341	2238	33.0	7	VI-1
12	Ipa_025897	24978156	24981612	F	gene	3457	2883	16.6	4	XI-1
12	Ipa_026259	30367258	30370348	R	gene	3091	918	70.3	4	XI-1	14
12	Ipa_026261.2	30385798	30387480	R	gene	1683	1593	5.3	2	XI-1	14
12	Ipa_026266	30460474	30461613	F	gene	1140	1140	0	1	Xb-1
12	Ipa_026299	30962119	30965653	F	gene	3535	3249	8.1	2	XI-1
12	Ipa_026406	32083236	32086544	R	gene	3309	3156	4.6	3	XI-1	15
12	Ipa_026408	32094665	32097486	R	gene	2822	1941	31.2	4	XI-1	15
12	Ipa_026576.2	34203565	34206647	F	gene	3083	2952	4.2	2	XI-1	16
12	Ipa_026578	34231790	34235177	F	gene	3388	2874	15.2	2	XI-1	16
12	Ipa_026679	35383695	35386367	R	gene	2673	2673	0	1	XI-1
12	Ipa_026715	35695693	35698338	F	gene	2646	1938	26.8	2	III
12	Ipa_026836	36933332	36938520	R	gene	5189	2589	50.1	4	XIV
12	Ipa_026899	37608933	37612337	F	gene	3405	3405	0	1	VII-3
12	Ipa_026946	38087889	38092580	R	gene	4692	2952	37.1	2	XI-1
12	Ipa_027140	39967165	39970775	F	gene	3611	2847	21.2	2	XI-1
13	Ipa_027578	18489477	18518414	F	gene	28938	2784	90.4	15	I-1
13	Ipa_027851	23803059	23803753	F	pseudo	695	498	28.3	3		17
13	Ipa_027855	23837840	23839328	F	gene	1489	1416	4.9	2	XI-1	17
13	Ipa_027856	23844155	23846623	F	gene	2469	2274	7.9	3	XI-1	17
13	Ipa_028343	29644340	29649199	F	gene	4860	1983	59.2	13	VI-2
13	Ipa_028442	30508206	30511434	F	gene	3229	2049	36.5	4	III
14	Ipa_028588	3927584	3948609	F	gene	21026	5322	74.7	27	VI-1
14	Ipa_029103	23484415	23487999	R	gene	3585	2721	24.1	5	Xb-2
14	Ipa_029408	27869414	27871330	F	gene	1917	1917	0	1	XI-2
14	Ipa_029465	28545549	28549420	F	gene	3872	2298	40.7	7	VI-1
14	Ipa_029646	31280721	31283351	F	gene	2631	2631	0	1	XII-1
14	Ipa_029896	33533177	33549855	R	gene	16679	3678	77.9	19	I-1	18
14	Ipa_029898	33563456	33587664	R	gene	24209	2346	90.3	15	I-1	18
15	Ipa_030245.2	1498010	1500780	F	gene	2771	2574	7.1	3	XI-1
15	Ipa_030305.2	2576708	2581185	F	gene	4478	1989	55.6	5	III
15	Ipa_030515.2	5818811	5822197	R	gene	3387	2892	14.6	2	IX
15	Ipa_030768.2	9753452	9759969	R	gene	6518	1593	75.6	14	II
15	Ipa_030792	10359000	10365568	F	gene	6569	2421	63.1	14	VIII-1
15	Ipa_031117	22283808	22298041	F	gene	14234	2739	80.8	15	I-1
15	Ipa_031150	22862868	22865195	R	gene	2328	2328	0	1	N2
15	Ipa_031443.2	28539418	28542993	R	gene	3576	3177	11.2	3	XII-1	19
15	Ipa_031443.N	28534629	28538436	F	gene	3808	3216	15.5	3	XII-1	19
15	Ipa_031521	29517844	29520526	R	gene	2683	2571	4.2	2	III
16	Ipa_032089.2	5215786	5219535	R	gene	3750	3051	18.6	2	XII-1
16	Ipa_032149.2	6096281	6099532	R	gene	3252	2784	14.4	3	XI-1	20
16	Ipa_032151	6132606	6137123	R	pseudo	4518	1002	77.8	3		20
16	Ipa_032165	6330142	6330702	R	pseudo	561	561	0	1		20
16	Ipa_032330	8913244	8916459	F	gene	3216	3111	3.3	2	XI-1	21
16	Ipa_032331	8947062	8950562	F	gene	3501	3123	10.8	3	XI-1	21
16	Ipa_032332	8972171	8976526	F	gene	4356	3531	18.9	3	XI-1	21
16	Ipa_032333	8998755	8999045	F	pseudo	291	291	0	1		21
16	Ipa_032384	9654607	9663889	R	gene	9283	1875	79.8	11	II
16	Ipa_032421.2	10303705	10309289	F	gene	5585	3696	33.8	14	XI-1	22
16	Ipa_032424	10357188	10363746	F	gene	6559	3561	45.7	12	XI-1	22
16	Ipa_032443	10731991	10736156	F	gene	4166	3120	25.1	2	XI-1	22
16	Ipa_032446	10772304	10772672	R	pseudo	369	369	0	1		22
16	Ipa_032447	10773258	10776137	R	gene	2880	2703	6.1	2	XI-1	22
16	Ipa_032493	11858924	11862876	F	gene	3953	3264	17.4	2	XI-1
17	Ipa_033063	11359613	11367472	R	gene	7860	1968	75.0	2	III
17	Ipa_033196	15217396	15220797	F	gene	3402	3402	0	1	Xb-1
17	Ipa_033286	16894832	16898857	F	gene	4026	2766	31.3	3	IX
17	Ipa_033301	17519116	17522955	F	gene	3840	2850	25.8	2	IX
17	Ipa_033436	20007808	20008904	F	pseudo	1097	939	14.4	2
17	Ipa_033585	22626185	22632509	F	gene	6325	2007	68.3	2	III
17	Ipa_033734	24416388	24418983	F	gene	2596	1242	52.2	11	II
17	Ipa_033993	26988975	26990070	R	pseudo	1096	936	14.6	2
17	Ipa_033994	26999099	27007603	R	gene	8505	1896	77.7	2	III
17	Ipa_034194	29069573	29078339	F	gene	8767	2034	76.8	18	V
17	Ipa_034336	30564643	30567163	F	gene	2521	1209	52.0	4	XI-2
18	Ipa_034636	11081119	11083980	F	gene	2862	2862	0	1	XII-1
18	Ipa_034847	16594224	16595929	R	gene	1706	1554	8.9	3	N1
18	Ipa_035209.2	21014947	21018573	R	gene	3627	2907	19.9	3	VII-1
18	Ipa_035293	21785534	21789844	F	gene	4311	3123	27.6	2	XI-1
18	Ipa_035545	24225804	24239701	F	gene	13898	2646	81.0	8	III
19	Ipa_036533	9288672	9290684	F	gene	2013	1896	5.8	2	Xa
19	Ipa_036587	10168172	10187528	R	gene	19357	3201	83.5	22	VIII-1
19	Ipa_036677	12226341	12237119	F	gene	10779	2151	80.0	9	VI-2
19	Ipa_036853	18946490	18972775	F	gene	26286	3615	86.2	29	VIII-1
20	Ipa_038176	16300007	16307219	R	gene	7213	2133	70.4	16	V
20	Ipa_038184.2	16422605	16428072	R	gene	5468	3225	41.0	2	Xb-1
20	Ipa_038196	16608848	16613637	F	gene	4790	3405	28.9	3	XI-1
20	Ipa_038252	17285276	17288147	R	gene	2872	1917	33.3	3	III
20	Ipa_038259	17437821	17450516	R	gene	12696	2151	83.1	16	V
20	Ipa_038296	17869857	17910456	R	gene	40600	2658	93.5	13	I-1	23
20	Ipa_038297	17933860	17934463	R	pseudo	604	360	40.4	3		23
20	Ipa_038574	21368431	21410922	R	gene	42492	1575	96.3	4	III	24
20	Ipa_038603	21802364	21807048	F	gene	4685	1956	58.2	3	III	24
20	Ipa_038674	22513065	22519599	R	gene	6535	1812	72.3	13	XIIIa
20	Ipa_038751	23404875	23407405	F	gene	2531	2004	20.8	2	III
20	Ipa_038796	23976957	23980388	R	gene	3432	3327	3.1	2	XI-1
20	Ipa_038846	24451378	24453447	F	gene	2070	2070	0	1	XI-2
20	Ipa_039022	26859652	26867653	R	gene	8002	1524	81.0	10	II
20	Ipa_039178	28968029	28971493	R	gene	3465	1917	44.7	2	III
21	Ipa_039266.2	2716238	2718410	F	gene	2173	1164	46.4	3	III
21	Ipa_039554	21867614	21916964	R	gene	49351	2679	94.6	23	IX
21	Ipa_039827	31812462	31817322	R	gene	4861	1983	59.2	13	VI-2
21	Ipa_040039	38585498	38591656	F	gene	6159	2889	53.1	19	VIII-1
21	Ipa_040322	44809764	44819474	R	pseudoP	9711	1008	89.6	6
21	Ipa_040324	44840866	44843337	F	gene	2472	2016	18.4	2	III
21	Ipa_040363.2	45387928	45391258	R	gene	3331	2847	14.5	2	XI-1
21	Ipa_040399	46569768	46572916	F	gene	3149	2784	11.6	4	VII-2
21	Ipa_040496	48096184	48098675	F	gene	2492	1902	23.7	2	III
21	Ipa_040737	52316917	52319960	R	gene	3044	2028	33.4	2	III
21	Ipa_040815	53913704	53915716	R	gene	2013	1896	5.8	2	Xa
21	Ipa_041094	58833089	58843535	R	gene	10447	1542	85.2	11	II
21	Ipa_041132	59596827	59599166	R	gene	2340	1629	30.4	3	III
21	Ipa_041473	66412019	66421384	R	pseudo	9366	951	89.8	4
21	Ipa_041490	66894278	66895016	F	pseudo	739	282	61.8	3
21	Ipa_041726	70753395	70753784	F	pseudo	390	231	40.8	3

• Ch = chromosome; D = direction (F = forward; R = reverse); pseudo = pseudogene; pseudo^P = possible pseudogene; nt = nucleotide; # = number; CDS = coding sequence; ¹ = gene data did not consider splice variants (AS); ² = intron length as percentage of gene length; ³ = clusters are formed considering genes of the same group which interval length do not exceed about 500000 nt.

Table 2
LRR-RLK genes groups and major features in yerba mate.

Group	Yerba mate				Arabidopsis				characteristic domains
	# genes	PML	PMW	PMI	# genes1	PML	PMW	PMI
I-1	10 (12)	868.6	96.36	6.56	48 (6)	824.7	92.15	6.56	SP|malectin|LRR|TM|K
I-2	1	421.0	46.97	7.42	2 (2)	392.8	43.80	8.79	TM|K
II	11 (11)	458.6	50.80	7.41	14 (4)	628.3	69.74	6.50	SP|LRR|TM|K
III	40 (18)	701.2	76.90	7.50	47 (5)	669.0	73.42	7.50	SP|LRR|TM|K3
IV	4 (6)	691.8	75.71	7.12	3	686.3	75.32	7.02	SP|LRR|TM|K
V	8 (16)	656.5	71.65	7.27	9 (4)	710.0	77.42	7.27	SP|LRR|TM|K
VI-1	4 (9)	763.2	84.56	8.56	5 (1)	758.0	83.75	8.36	SP|LRR|TM|K
VI-2	8 (11)	622.3	68.45	6.17	8 (6)	564.0	62.92	6.60	SP|LRR|TM|K
VII-1	4	930.0	101.13	6.44	5	943.8	103.12	6.51	SP|LRR|TM|K
VII-2	4 (5)	846.6	93.47	7.03	3	768.0	84.87	7.81	SP|LRR|TM|K
VII-3	2	1135.5	123.48	5.90	2	1138.0	123.60	7.12	SP|LRR|TM|K
VIII-1	11 (12)	930.7	102.04	6.76	8	918.6	101.67	6.85	SP|LRR|TM|K
IX	18 (12)	913.7	99.95	6.38	16 (5)2	1001.1	110.51	7.03	SP|LRR|malectin|TM|K4
Xa	4	617.8	67.76	7.45	4	604.3	66.77	7.26	SP|LRR|TM|K
Xb-1	10 (2)	1047.3	114.48	6.21	9 (1)	1052.5	115.62	6.45	SP|LRR|TM|K
Xb-2	2	995.5	109.00	5.58	1	1106.0	121.90	8.85	SP|LRR|TM|K
XI-1	59 (27)	979.5	107.44	6.64	34 (4)	1032.1	113.49	6.39	SP|LRR|TM|K
XI-2	3 (2)	503.8	56.35	9.35	2 (2)	431.0	48.00	9.74	SP|LRR|TM|K
XII-1	10	1028.7	112.51	7.05	8	967.6	106.28	7.10	SP|LRR|TM|K
XIIIa	2 (2)	344.3	38.72	8.74	4 (2)	553.3	61.03	6.57	SP|LRR|TM|K
XIIIb	2 (1)	889.0	97.85	6.24	3 (1)	956.8	105.49	6.72	SP|LRR|TM|K
XIV	3 (6)	766.0	83.84	7.94	2 (1)	824.3	90.35	9.39	SP|LRR|TM|K
XV	4	1116.5	122.16	6.78	2	845.5	92.49	6.76	SP|LRR|TM|K
N1	1 (1)	475.5	54.78	7.43	1	518.0	59.73	7.38	SP|LRR|TM|K
N2	1	775.0	83.71	6.75	1	776.0	85.30	5.69	SP|RCC1/BLIP-II|TM|K
Total	226 (153)				241 (44)
Mean		779.1	85.60	7.07		786.8	86.76	7.29	SP|LRR|TM|K

• # genes = number of genes (isoforms); PML = protein mean length (nt); PMW = protein mean molecular weight (kDA); PMI = protein mean isoelectric point; SP = signal peptide; ¹ = five genes hold short proteins with solely K domain; ² = without considering short protein AT4G29654 that grouped into VII-3; ³ = Ipa_004205 holds PF00179 domain also; ⁴ = half yerba mate genes lack malectin domain.

We also found three possible pseudogenes containing a non-cytoplasmatic domain in the corresponding region to the LRR domain, in addition to TM and complete kinase domains (Fig. 1d-e; Tab. S1; File S2). Two of them were similar to yerba mate active LRR-RLK genes, and the other was comparable to an inactive LRR-RLK gene from other species (File S3). Additionally, a signal peptide was commonly found at the N-terminus of LRR-RLK proteins (Tab. 2; Fig. 1c), as expected for such membrane-integrated molecules.

When considering the 226 LRR-RLK genes of yerba mate, the distribution of values for gene structural parameters was highly dispersed (Tab. 1; Fig. 1f). These parameters included gene length (ranging from 1,140 to 55,593 nt; mean: 7,273.5 nt), total CDS length (915 to 5,322 nt; mean: 2,607.7 nt), total intron length (0 to 52,653 nt; mean: 4,665.8 nt), and the number of coding exons (1 to 31; mean: 6.9). Among these, the CDS length exhibited the most moderate distribution (Fig. 1f). Intron-less genes accounted for 10.6% (24 genes). In contrast, the remaining 202 genes contained introns that spanned between 1.7% and 96.3% of the gene length (mean: 44.9%) (Tab. 1). Furthermore, 67.7% (153 genes) had a low number of exons (1-5): 1 (24 genes), 2 (70 genes), 3 (26 genes), 4 (19 genes), or 5 (14 genes) exons per gene (Tab. 1). The 20 identified (possible) pseudogenes showed consistently lower structural values (Tab. 1): gene length (4,011 nt), total CDS length (783.3 nt), total intron length (3,228.1 nt), and number of coding exons (2.7).

We constructed an unrooted Maximum Likelihood (ML) phylogenetic tree for the LRR-RLK genes of yerba mate and A. thaliana (Fig. 2a; Files S4-S5). The tree resolved 25 highly supported groups (labeled I to XV, N1, and N2; Tabs. 1, S3), grouping homologous proteins from both species. The major features of these groups are detailed in Table 2. Within groups, a strong correlation was observed between yerba mate and A. thaliana regarding the mean protein length, molecular weight, and isoelectric point, in addition to shared characteristic domains and gene count (Table 2). Groups I-1 and XI-1 were notable for exhibiting a markedly divergent gene expansion between the two species (Tab. S1). Groups XI-1 (59 genes), III (40 genes), and IX (18 genes) were the largest, comprising over half of the species’ LRR-RLK clade (Fig. 2b; Tab. 2). Furthermore, 58 neighbor genes from the same phylogenetic group, along with 8 pseudogenes, were distributed into 24 clusters across 12 chromosomes (Tab. 1; Figs. 1d, 2c), with group XI-1 accounting for half of these (Fig. 2d).

Figure 2
a-d. Characterization of LRR-RLK genes in yerba mate - a. unrooted Maximum Likelihood (ML) phylogenetic tree of LRR-RLK genes of yerba mate and A. thaliana. Each of the 25 groups includes homologous proteins from both species. Values in brackets denote support values for each group; b. distribution of the 226 LRR-RLK genes into the 25 phylogenetic groups; c. distribution of the 58 clustered LRR-RLK genes (and 8 pseudogenes) per chromosome; d. distribution of the 58 clustered LRR-RLK genes per phylogenetic group.

GO annotation identified 677 GO Full Term IDs, associated with 173 genes and the possible pseudogene Ipa_40322 (Tab. S4). Subsequent mapping collapsed these terms into 35 GO Slim Term IDs and Labels (Tab. S5). The major GO Slim categories describing the molecular function (MF), biological process (BP), and cellular component (CC) in which these genes are involved included (Fig. 3a): MF: transferase activity (172 genes), catalytic activity, acting on a protein (172 genes), molecular transducer activity (49 genes); BP: signaling (51 genes), anatomical structure development (41 genes), reproductive process (26 genes), cell differentiation (12 genes), defense response to other organism (12 genes); CC: plasma membrane (149 genes).

Figure 3
a-c. Characterization of LRR-RLK genes in yerba mate - a. distribution of LRR-RLK genes into GO Slim Term Labels; b. distribution of LRR-RLK proteins into predicted subcellular compartments; c. distribution of LRR-RLK proteins into subcellular compartments, considering only the best WoLF PSORT rank per gene.

Subcellular localization analysis with WoLF PSORT (Tab. S6), predicted five major compartments associated with the 226 genes and three possible pseudogenes (Fig. 3b): plasma membrane (197 genes), vacuolar membrane (151 genes), endoplasmic reticulum (127 genes), chloroplast (92 genes) and nuclear (91 genes) (Fig. 3b). When considering only the best WoLF PSORT prediction rank per gene, the plasma membrane (129 genes) and chloroplast (55 genes) were the locations for 80.3% of LRR-RLK proteins (Fig. 3c).

Five-prime and 3-prime promoter analysis of the 226-yerba mate LRR-RLK genes and three possible pseudogenes revealed a comparable pattern of binding sites for 26 exclusive Transcription Factor (TF) families (Tab. 3; Tab. S7-S12; Fig. 4a,c; Files S6-S7). Furthermore, both promoter regions were dominated by the same 10 TF families, which accounted for 88.4% of binding sites: Dof (20.7%), BBR-BPC (10.1%), MIKC_MADS (10.0%), AP2 (9.9%), GRAS (8.4%), ERF (6.5%), C2H2 (6.2%), TALE (5.8%), CPP (5.7%), and HD-ZIP (5.1%). Regarding Transcription Factor Family Binding Sites (TFFBS) stringency (defined as TFFBS ≥10%, 5%, or 2% of the promoter length), Dof showed a slightly superior presence in 5-prime regions (179, 224, and 228 genes, respectively) compared to 3-prime regions (134, 208, and 224 genes, respectively) (Tab. 3). At 2% stringency, the TF families AP2, MIKC_MADS, BBR-BPC, and GRAS were present in approximately 200 genes across both the 5-prime and 3-prime regions (Tab. 3). At the same stringency level, HD-ZIP, C2H2, TALE, ERF, and CPP populated 5-prime regions of an average of 179 genes and 3-prime regions of an average of 133 genes (Tab. 3). Finally, at 5% stringency, 5-prime regions of 133 genes (58.0%) hosted 4-7 distinct TF families, whereas 3-prime regions of 142 genes (62.0%) were occupied by 2-5 distinct TF families (Fig. 4b,d).

Table 3
Binding sites to families of TFs at promoter regions of LRR-LRK genes of yerba mate.

TFFBS	# genes	# nt	% nt	# genes	# genes	# genes
				TFFBS ≥10%	TFFBS ≥5%	TFFBS ≥2%
2000 nucleotides 5-prime promoter sequences
Dof	229	68700	19.621	179	224	228
AP2	225	33235	9.492	52	171	216
MIKC_MADS	227	33045	9.438	50	165	217
BBR-BPC	223	36390	10.393	64	164	211
GRAS	224	28559	8.157	30	141	211
HD-ZIP	215	21431	6.121	17	93	185
C2H2	209	21020	6.003	21	83	176
TALE	212	19659	5.615	14	80	183
ERF	210	23661	6.758	32	77	173
CPP	221	20065	5.731	15	71	176
MYB	189	10602	3.028	4	22	95
LBD	124	8103	2.314	9	21	64
GATA	111	6132	1.751	7	14	41
B3	201	9249	2.642	-	12	96
MYB-related	72	2805	0.801	-	6	23
Trihelix	118	2683	0.766	-	-	4
TCP	57	1745	0.498	-	-	11
C3H	100	2161	0.617	-	-	15
ARF	1	21	0.006	-	-	-
bHLH	10	176	0.050	-	-	-
FAR1	1	12	0.003	-	-	-
G2-like	4	75	0.021	-	-	-
NAC	7	123	0.035	-	-	-
Nin-like	3	48	0.014	-	-	-
SBP	13	224	0.064	-	-	-
WRKY	16	208	0.059	-	-	-
Total	229	350132	100.000	194	228	229
2000 nucleotides 3-prime promoter sequences
Dof	227	52714	21.715	134	208	224
MIKC_MADS	223	25583	10.539	20	123	214
AP2	222	25140	10.356	16	122	211
BBR-BPC	212	23952	9.867	23	114	187
GRAS	221	20945	8.628	8	95	196
C2H2	207	15703	6.469	8	53	143
ERF	192	14931	6.151	10	46	134
TALE	195	14408	5.935	4	51	155
CPP	215	13889	5.721	6	38	124
HD-ZIP	178	9991	4.116	3	22	107
B3	179	7366	3.034	-	7	74
MYB	143	6074	2.502	2	8	49
LBD	83	3363	1.385	2	4	30
TCP	59	2220	0.914	-	2	14
GATA	60	1942	0.800	1	3	9
C3H	82	1424	0.587	-	-	3
MYB-related	43	1168	0.481	-	1	5
Trihelix	42	770	0.317	-	-	-
bHLH	16	342	0.141	-	-	2
ARF	15	320	0.132	-	-	-
NAC	12	213	0.088	-	-	-
SBP	6	96	0.040	-	-	-
G2-like	5	74	0.030	-	-	-
Nin-like	4	64	0.026	-	-	-
WRKY	3	41	0.017	-	-	-
FAR1	2	24	0.010	-	-	-
Total	228	242757	100.000	141	220	227

• TFFBS = transcription factor family binding site; # genes = number of genes (including 3 possible pseudogenes); # nt = number of nucleotides; % nt = percentage of nucleotides considering total number of nucleotides; # genes TFFBS ≥10%, 5%, or 2% = number of genes which TFFBS are equal or superior than 10%, 5% or 2% considering 2000 nucleotides promoter sequences, respectively.

Figure 4
a-d. Characterization of LRR-RLK genes in yerba mate - a, c. percentage distribution of Transcription Factor (TF) binding sites in the 5-prime (a) and 3-prime (c) promoter regions of the LRR-RLK genes; b, d. number of LRR-RLK genes vs. number of Transcription Factor families (fam.) per gene with binding site length ≥5% of promoter length.

The expression pattern of the 226-yerba mate LRR-RLK genes and three possible pseudogenes was further investigated using 26 distinct RNA-seq libraries (Tab. S1,S13). These libraries were derived from leaves, roots, and a plantlet, representing two major cultivars (Pg538-INTA and SI-49) and five other individual plants sourced from Misiones and Corrientes, Argentina. Based on their TPM values, all 229 LRR-RLK genes and possible pseudogenes exhibited detectable expression across all distinct libraries and tissues analyzed (File S8; Tab. S13). To further classify their expression profiles, a heatmap and dendrogram were constructed (Fig. 5; Fig. S1). Notably, 20 genes showed high and comparable expression levels across all tissues and conditions: Ipa_033734, Ipa_036587, Ipa_020099, Ipa_022765, Ipa_038846, Ipa_030515.2, Ipa_038176, Ipa_039022, Ipa_027578, Ipa_025666, Ipa_000278, Ipa_029896, Ipa_040815, Ipa_036533, Ipa_036853, Ipa_005844, Ipa_035545, Ipa_031117, Ipa_041094, and Ipa_026266. It is also worth noting that half of the total genes exhibited lower and comparable expression levels across all tissues and conditions.

Figure 5
Heatmap and dendrogram of the expression profiles (based on TPM values) of the LRR-RLK genes of yerba mate across diverse RNA-seq libraries (1-26) - 1 = SRR3584948; 2 = SRR1411087; 3 = SRR3584951; 4 = SRR3584952; 5 = SRR3584957; 6 = SRR3584945; 7 = SRR3584956; 8 = SRR3584954; 9 = SRR3584955; 10 = SRR3584947; 11 = SRR3584946; 12 = SRR5740154; 13 = SRR5740155; 14 = SRR5740159; 15 = SRR5740157; 16 = SRR5740156; 17 = SRR5740158; 18 = SRR24273722; 19 = SRR24273721; 20 = SRR24273720; 21 = SRR24273719; 22 = SRR24273718; 23 = SRR24273717; 24 = SRR3584943; 25 = SRR3584944; 26 = SRR3584950.

To determine the differential expression (DE) of the 229 LRR-RLK genes and possible pseudogenes of yerba mate, DESeq2 analysis was performed on the species’ overall gene set, considering diverse experimental conditions (File S9; Tab. S14). The assayed conditions were: 1. Effect of Disease in Leaves: Curly vs. Normal; 2. Effect of Drought Stress in Leaves: Stressed vs. Untreated; 3. Effect of Drought Stress in Roots: Stressed vs. Untreated; 4. Effect of Tissue: Normal Leaves vs. Normal Roots. For all conditions, quality control parameters, such as the principal component of the variance, sample-to-sample distances, dispersion of normalized counts, p-value frequency, and log fold change (volcano plot), were deemed adequate to support the conclusions (File S9). A heatmap was constructed to visualize the fold change of the differentially expressed genes (Fig. 6). Across all assays, a total of 220 genes and possible pseudogenes (96.1%) of yerba mate were significantly affected (induced or repressed) by at least one condition. These affected genes showed varied responses across conditions: four conditions: 67 genes (30.5% of affected genes); three conditions: 81 genes (36.8%); two conditions: 53 genes (24.1%); one condition: 19 genes (8.6%). Specific responses for each condition included: disease in leaves affected 121 LRR-RLK genes (91 downregulated vs. 30 upregulated); drought stress in leaves affected 160 genes (91 downregulated vs. 69 upregulated); drought stress in roots affected 163 genes, predominantly repressed (137 downregulated vs. 26 upregulated); tissue comparison (leaves vs. roots) revealed 192 LRR-RLK genes with significant DE, with 149 genes showing higher expression in roots, and 43 genes with higher expression in leaves.

Figure 6
Differential Expression (DE) analysis graph of LRR-RLK genes of yerba mate considering diverse experimental conditions (A-D). Conditions: (A) Effect of disease in leaves (Curly vs. Normal); (B) Effect of drought stress in leaves (Stressed vs. Untreated); (C) Effect of drought stress in roots (Stressed vs. Untreated); (D) Effect of tissue (Normal leaves vs. Normal roots). Legend: SU = Strongly Upregulated: green; MU = Moderately Upregulated: pea green; LU = Lightly Upregulated: light green; SD = Strongly Downregulated: red; MD = Moderately Downregulated: orange; LD = Lightly Downregulated: light orange. White squares denote a non-significant FDR-adjusted p-value (> 0.05).

Discussion

The number of kinase genes found in yerba mate (1,200) aligned with the mean of 1,403±752 kinase genes per species across 197 plant species from the iTAK database (Zheng et al. 2016). Moreover, a similar kinase landscape was observed between yerba mate and A. thaliana, considering the 1,028 kinase genes, the size of the RLK-Pelle family (61.3% of overall kinase genes), and the size of the LRR-RLK subfamilies (36.4% of RLK-Pelle family genes) identified in the latter species by iTAK (Zheng et al. 2016). This structural similarity enabled a well-supported comparison between both species.

Recent research on LRR-RLK genes in plants has centered on their fundamental roles as membrane-bound sensors. Studies have focused on how these genes balance plant growth and development, innate immunity (detecting pathogen patterns), and stress adaptation (cold, drought, and salt stress) (Da Silva Dambroz et al. 2023; Yan et al. 2023; Li et al. 2025; Sampaio et al. 2025; Zhiqi et al. 2025).

To further characterize the LRR-RLK gene repertoire in yerba mate, we utilized its recently assembled and annotated chromosome-level nuclear genome (Aguilera & Grabiele 2025). The identification of 226 LRR-RLK genes distributed across the 20 chromosomes of the yerba mate genome highlights a significant molecular repertoire dedicated to environmental sensing and developmental coordination (Man et al. 2020) in this crop species. A comparable LRR-RLK gene count was found in A. thaliana (241; Yin et al. 2024; Zheng et al. 2016) and tomato (234; Wei et al. 2015), despite yerba mate holding a significantly larger genome (~872.9 Mb) and a much higher total protein-coding gene count (~41.9k) than those species (Aguilera & Grabiele 2025). This discrepancy suggests, a priori, a more stringent stabilizing selection to maintain signaling homeostasis in I. paraguariensis. Furthermore, the numerous alternative splice variants identified for LRR-RLKs in yerba mate (153) contribute to enhancing the proteomic diversity of these receptors, a common strategy in plants to fine-tune responses to environmental fluctuations within the constraints of the existing genome or clade size (Aballay et al. 2026).

The LRR-RLK proteins in yerba mate were found to be comparable to those of other plants regarding the typical arrangement of LRR, TM, and kinase domains, as well as the vast diversity in the number and arrangement of LRR motifs that ultimately aid in perceiving a wide array of extracellular signals (Bezerra-Neto et al. 2020; Li et al. 2018). At the nucleotide level, the LRR-RLK genes of yerba mate showed high dispersion in structural parameters, such as gene length, total intron length, and the number of coding exons, reflecting a dynamic genome that aligns with previous reports in plants (Liu et al. 2017). In contrast, the CDS length of these LRR-RLK genes appeared more constrained, in agreement with the structural resemblance observed at the protein level between yerba mate and other plant species.

Additionally, the 10.6% of intron-less LRR-RLKs in yerba mate are noteworthy, as genes that do not require splicing are often prioritized for rapid transcription during stress responses (Jeffares et al. 2008). On the other hand, the identification of 20 LRR-RLK pseudogenes displaying complete or partial kinase domains, many of which neighbor active genes, agrees with the common birth-and-death model of gene evolution. While pseudogenes are frequently dismissed as non-functional, recent studies suggest that certain receptors retaining a kinase domain may serve as decoys that modulate intracellular signaling (Anbu et al. 2023). In addition, approximately 27% of the LRR-RLK genes and pseudogenes in yerba mate are distributed across 24 clusters spanning 12 chromosomes, supporting the hypothesis that tandem duplications are key drivers for the diversification of these receptors, as has been reported in several plant lineages (Panchy et al. 2016). Notably, tandem duplications are associated with the massive expansion of LRR-RLKs involved in stress responses, whereas developmental subfamilies typically lack this evolutionary signature (Kileeg et al. 2023).

The classification of yerba mate LRR-RLKs into 25 homology groups mirrors the Arabidopsis system and remains consistent across plant lineages (Lehti-Shiu & Shiu 2012; Yin et al. 2024; Zheng et al. 2016). Groups I-1 and XI-1 were notable for exhibiting a markedly divergent gene expansion between yerba mate and A. thaliana, which may indicate specialized evolutionary trajectories. More than half of the LRR-RLK genes in yerba mate are distributed into three main groups: XI-1 (59 genes, half of which are clustered), III (40 genes), and IX (18 genes), whereas these same groups encompass only 40% of the receptors in A. thaliana (Yin et al. 2024; Zheng et al. 2016). For instance, proteins from groups I-1 and IX are unique in exhibiting an internal malectin domain, which in Arabidopsis is associated with controlling growth, reproduction, and stress responses (Yang et al. 2021).

Combined GO annotation and subcellular localization analysis of the yerba mate LRR-RLK proteins confirmed that 96.9% of these receptors are primarily located in the plasma membrane, as expected (Man et al. 2020). Secondary assignment to compartments other than the plasma membrane occurred in yerba mate and has also been reported in disparate plants as kiwifruit (Wang et al. 2025) and pine (Nie et al. 2022). These findings likely reflect the continuous intracellular trafficking of LRR-RLK proteins. In this regard, the overlap of targeting signals has been reported for kinases that re-localize from the plasma membrane to the chloroplasts to promote chloroplast-mediated defense responses (Medina-Puche et al. 2020), supporting our findings regarding LRR-RLK proteins in yerba mate. In addition, GO annotation assigned 75.1% of the yerba mate LRR-RLK proteins to the category of molecular transducer activity sensu lato, even though 100% of these genes possess complete kinase domains responsible for signal transduction. Remarkably, ontology analysis also associated 18.8% of yerba mate LRR-RLKs with developmental processes and linked 5.2% to the immune system.

Analysis of the promoter regions located upstream and downstream of the 226 LRR-RLK genes and three potential pseudogenes of yerba mate revealed a comparable pattern of binding sites for 26 distinct TF families. Furthermore, both promoter regions were dominated by the same 10 TF families, which collectively accounted for 88.4% of the total binding sites: Dof, BBR-BPC, MIKC_MADS, AP2, GRAS, ERF, C2H2, TALE, CPP, and HD-ZIP. This remarkable similarity between the upstream and downstream regions of LRR-RLKs appears to be adaptive, especially when considering the 58 TF families described for yerba mate (Aguilera & Grabiele 2025). Under a TFFBS stringency threshold of ≥2%, Dof, AP2, MIKC_MADS, BBR-BPC, and GRAS sites dominated the promoter regions of 200 or more LRR-RLKs in yerba mate. Collectively, these TF families have been described as key regulators of various vital aspects of plant life. Dof genes regulate hormone signaling, diverse biotic and abiotic stress responses, dormancy, tissue differentiation, carbon and nitrogen assimilation, and carbohydrate metabolism (Zou & Sun 2023), while AP2 family members are linked to vegetative and reproductive organ development (Zhao et al. 2019). MIKC_MADS TFs are associated with seed germination, vegetative growth, the transition to flowering, and flower development and senescence (Lai et al. 2019), whereas homeotic gene regulation, developmental processes, hormone signaling, stress responses, circadian oscillations, and sex determination activities have been linked to the BBR-BPC family (Sahu et al. 2023). Finally, GRAS genes regulate gibberellic acid and brassinosteroid signaling, development, phytochrome signal transduction, nodulation and arbuscular mycorrhizal symbiosis, and abiotic stress responses (Jaiswal et al. 2022). In summary, promoter analysis indicates that 200 or more LRR-RLK genes in yerba mate are heavily associated with developmental processes and stress responses.

Regarding their expression profiles, the 226-yerba mate LRR-RLK genes and three potential pseudogenes exhibited detectable expression across all analyzed libraries and tissues. Remarkably, a core of nearly 59% of these genes was found to be constitutively highly or lowly expressed, suggesting a basal regulatory role in maintaining essential growth processes. Subsequent differential expression (DE) analysis revealed that 96.1% of the yerba mate LRR-RLK genes were significantly induced or repressed under at least one assayed condition. In leaves affected by curly leaf disease, 39.7% of LRR-RLK genes were downregulated and 13.1% were upregulated. Leaf curling is a symptom of a widespread disease caused by the psyllid Gyropsylla spegazziniana (Lizer 1919), a major pest of yerba mate that induces leaf deformations and causes significant economic losses in this crop (Candia et al. 2026). Additionally, drought-stressed leaves showed 39.7% downregulation and 30.1% upregulation of these receptors, whereas drought-stressed roots displayed 59.8% downregulated and 11.4% upregulated genes, highlighting tissue-specific differences in LRR-RLK expression profiles. These differences became more pronounced in the DE tissue comparison, where 65% of the receptors showed higher expression in roots, while another 18.8% exhibited higher expression in leaves.

Overall, LRR-RLK genes were primarily downregulated (ranging from 39.7% to 59.8%) in curly leaf disease-affected leaves as well as drought-stressed leaves and roots. Massive downregulation of LRR-RLKs has also been documented in several plant species affected by pathogens or drought stress, including tea (Zhang et al. 2024), cucumber (Yu et al. 2022), common bean (Da Silva Dambroz et al. 2023), and kiwifruit (Li et al. 2025). While the pathogens causing curly leaf disease may suppress host immunity, the robust downregulation under drought stress likely represents a resource-conservation strategy during water scarcity, suppressing genes related to growth and metabolic activities. Conversely, the upregulated LRR-RLK receptors (11.4%-30.1%) that respond strongly to drought and curly leaf disease constitute promising candidate genes for selection-mediated improvement. The comprehensive mapping of these genes onto the 20 chromosomes provides a valuable baseline for precision breeding, enabling the development of yerba mate cultivars that maintain high productivity while thriving under the increasing environmental challenges of modern cultivation systems.

Acknowledgements

We are grateful to the Galaxy teams for making their servers and bioinformatic tools openly available. We also want to thank to the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina), where we are both associate researchers, for continuous support. We dedicate this work to our beloved Lola and Serafin.

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Data availability statement

In accordance with Open Science communication practices, the authors inform that the genome assembly and annotation data analyzed in this study are publicly available under accession OMIX007912 in the NGDC BioProject PRJCA032295. The updated annotation of YMM1 containing the remodeled LRR-RLK genes is available in Mendeley Data under DOI 10.17632/pfg77fdctp.1. Supplementary tables and files are provided with the article." to Supplementary tables, figure and files are available in Figshare under <https://doi.org/10.6084/m9.figshare.32306913>.

⁴ Author for correspondence:

[e-mail]; [e-mail]

Area Editor:

Dr. Paulo Guimarães

Publication Dates

• Publication in this collection
  02 Nov 2026
• Date of issue
  2026

History

• Received
  18 May 2026
• Accepted
  06 July 2026