Transcriptional control of cuticular wax biosynthesis is important for plant adaptation to environmental stress. Although several transcription factors have been identified as key regulators of plant wax biosynthesis, the role of HD-ZIP III family members remains uncharacterized. Here, we demonstrate that the HD-ZIP III transcription factor PHABULOSA (CsPHB) positively regulates wax accumulation and cuticle barrier function in cucumber (Cucumis sativus L.). A gain-of-function mutation in CsPHB significantly increased the deposition of epicuticular wax crystals, resulting in greater cuticle thickness, lower cuticle permeability, and improved water retention capacity. Functional analyses of overexpression and CRISPR/Cas9-edited lines confirmed the role of CsPHB in regulating cuticular wax biosynthesis. Transcriptome profiling, promoter activation assays, and DNA-binding experiments further revealed that CsPHB directly activates fatty acyl-CoA reductase3-like (CsFAR3-like), which encodes a fatty acyl-CoA reductase participating in the primary alcohol biosynthesis pathway. Notably, CsFAR3-like was identified as a FAR family member that possesses primary alcohol-producing activity in cucurbits. Overexpression of CsFAR3-like led to greater wax accumulation and enhanced water retention, whereas knockout CsFAR3-like lines displayed lower wax accumulation and impaired cuticle function. Analysis of CsPHB gain-of-function × CsFAR3-like knockout double mutants revealed intermediate CsFAR3-like expression, wax accumulation, and water-deficit tolerance compared with the respective single mutants. Collectively, our findings uncover a previously uncharacterized CsPHB-CsFAR3-like transcriptional module that controls wax biosynthesis and cuticle function in cucumber, advancing our understanding of epidermal water retention mechanisms in this species.

Fatty acyl-CoA reductases (FARs) catalyze the formation of primary alcohols, which are key components of plant cuticular wax. Although a few FAR members have been functionally characterized in Arabidopsis and several other species, their roles remain largely unexplored in Cucurbitaceae crops. Here, we identified five CsFAR genes in the cucumber genome, unevenly distributed across chromosomes 1, 4, and 6. Phylogenetic and collinearity analyses revealed that CsFARs share conserved evolutionary relationships with FARs from Arabidopsis thaliana, wheat, and other Cucurbitaceae species. Promoter cis-element analysis indicated potential regulation of CsFARs by light, phytohormones, and multiple abiotic stresses. Among the five CsFAR members, CsFAR3-like exhibited the highest expression across tissues and was strongly induced by drought, salt, jasmonic acid, and Podosphaera xanthii infection. Subcellular localization confirmed its targeting to the endoplasmic reticulum and yeast expression validated its enzymatic activity in primary alcohol biosynthesis. Heterologous overexpression of CsFAR3-like in Arabidopsis enhanced cuticular wax deposition, reduced water loss, and improved drought tolerance. Furthermore, a canonical MYB-binding site was identified in the CsFAR3-like promoter, and dual-luciferase and yeast one-hybrid assays demonstrated that CsMYB96 directly binds and activates its transcription. Heterologous overexpression of CsMYB96 in Arabidopsis increased total wax content and improved drought tolerance. Together, these findings raise the possibility of a MYB96-FAR regulatory interaction involving CsMYB96 and CsFAR3-like and imply that CsFAR3-like might be involved in cucumber cuticular wax biosynthesis and stress responses. These data provide tentative mechanistic clues for future work on improving drought resilience in cucurbit crops.

The neuroactive β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) was first identified in Lathyrus sativus and present also in several Chinese traditional herbs including Panax notoginseng. It exhibit toxicological effects as the causative agent of neurolathyrism when L. sativus was over-consumed under drought-triggered famines or pharmacological effects including neuroprotection and wound healing. Determinating of β-ODAP synthetase (BOS) will accelerate plant improvement and utilisation of those species containing β-ODAP. In this report, trace level of β-ODAP was confirmed in several cultivars of Pisum sativum, a close relative of L. sativus. Functions of LsBAHD3 and LsAAE3 were investigated via its transient expression in Nicotiana benthamiana, in vitro enzymatic activity assay and overexpression in hairy roots of L. sativus and P. sativum, etc. The results suggested that LsBAHD3 act as BOS, while LsAAE3 function as oxalyl-CoA synthetase to catalyse/promote β-ODAP biosynthesis. Further comparison and verification of LsBAHD3-specific and LsAAE3-specific protein interactome suggested that the LsBAHD3-LsAAE3 module catalyses β-ODAP biosynthesis, and the ubiquitin/26S proteasome system is highly involved in the regulation of BOS and β-ODAP content and may be responsible for the different level of β-ODAP in L. sativus and P. sativum. These results provide valuable insight into the biochemical and genetic mechanisms of β-ODAP biosynthesis.

The presence of toxic heavy metals lead (Pb) and cadmium (Cd) in polluted soil damage crop production and consequently harms human and livestock health. Tartary buckwheat (Fagopyrum tataricum) is a potential model plant for heavy metal phytoremediation because of its valuable characteristics of high heavy metal tolerance and abundant biomass production. Here, we report that the Tartary buckwheat FtMYB46-FtNRAMP3 module enhances plant Pb and Cd tolerance. RNA sequencing analysis showed that Pb treatment specifically induced expression of FtNRAMP3, a member of the NRAMP (Natural Resistance-Associated Macrophage Protein) transporter gene family. Further cytological and biochemical analysis revealed that FtNRAMP3 was localised to the plasma membrane and significantly contributed to increased tolerance to Pb and Cd in yeast cells. Consistently, transgenic overexpression of FtNRAMP3 in Arabidopsis significantly increased plant tolerance to Pb and Cd applications, reducing Pb concentration but increasing Cd concentration in the overexpression transgenic plants. Subsequent yeast one-hybrid and electrophoretic mobility shift assays showed that the transcription factor FtMYB46 directly binds to the FtNRAMP3 promoter. Further, FtMYB46 promoted FtNRAMP3 expression and increased plant Pb and Cd tolerance. Overall, this study demonstrates the important role of the FtMYB46-FtNRAMP3 module and its potential value in the phytoremediation of Pb and Cd stress.

Novel antibiotics to combat global antimicrobial resistance (AMR) in human and animal pathogens are urgently required [[1][1]]. Antimicrobial peptides (AMPs) are a class of small molecules inhibiting growth of various microorganism, including both Gram-negative and Gram-positive bacteria, fungi and viruses [[2][2]]. These peptides are valued for their broad-spectrum antimicrobial activity, achieved through mechanisms such as bacterial membrane disruption or interference with intracellular processes rather than targeting specific proteins, which makes lower propensity to induce resistance. While AMPs have been extensively identified and verified from animal proteomes, reference microbial genomes and host environments, those from extreme environments remain unexplored. ### Competing Interest Statement The authors have declared no competing interest. National Key R&D Program of China, 2023YFA1800900, 2021YFA0910500, 2018YFC0910502 National Natural Science Foundation of China, 22574058, 32571640 [1]: #ref-1 [2]: #ref-2

Map-based cloning revealed that the mutation in a highly conserved amino acid of the CsPBGD, which encodes porphobilinogen deaminase, causes the phenotype of leaf necrosis and enhanced resistance to powdery mildew and gray mold in cucumber. Lesion mimic mutants (LMMs) are valuable genetic resources for studying programmed cell death (PCD) and disease resistance. Although a number of genes controlling lesion mimic have been identified in model species, none have been mapped or cloned in cucumber. Here, we identified two cucumber mutants, C1173 and C2123, which exhibit leaf necrosis due to PCD. Genetic analysis revealed that these phenotypes are controlled by two semi-dominant loci, ln1 and ln2, respectively. Both mutants were heterozygous, as homozygous dominants were lethal (one caused cotyledon etiolation lethality; the other was unobtainable). Fine mapping placed the ln1 locus within a 54.1 kb region on chromosome 3. Further investigation revealed ln1 and ln2 were allelic mutations, with CsPBGD (CsaV3_3G031800), encoding porphobilinogen deaminase, identified as the candidate gene for both mutants. Mutations in CsPBGD resulted in amino acid substitutions, Ala314Val in ln1 and Arg197Lys in ln2, disrupting enzyme activity and altering H₂O₂ accumulation. CsPBGD expression was significantly reduced in various organs of ln1. VIGS of CsPBGD in both cucumber and tobacco successfully displayed the leaf necrosis phenotype. CsPBGD proteins from both mutants and wild type (WT) were localized in chloroplasts. The mutants exhibited significantly enhanced resistance to powdery mildew (Podosphaera xanthii) and gray mold disease (Botrytis cinerea). Further studies showed that CsPBGD expression in the mutant was significantly more downregulated than in WT after P. xanthii infection, alongside increased H₂O₂ accumulation. This study is the first to characterize and clone CsPBGD in cucumber, revealing its involvement in resistance to disease.

BACKGROUND:A major allergen Fag t 2 in Tartary buckwheat is abundant in grains and has the potential to improve drought and disease resistance of Arabidopsis thaliana, which led to the hypothesis that the promoter of Fag t 2 had the function of regulating seed-specific expression and responding to stress. RESULTS:This study successfully cloned the Fag t 2 promoter using genome walking. The functions of the Fag t 2 promoter were investigated through gene gun bombardment, GUS staining and activity detection. The findings revealed that full-length promoter acted as a seed-specific promoter. As seed-specific elements decrease, the seed expression specificity diminishes progressively. The full-length and truncated promoters exhibited differential responses to phytohormone. However, the responsiveness of promoters varied between the protoplast and A. thaliana, suggesting that the regulatory function of the Fag t 2 promoter in stable transformation system is more complicated. CONCLUSION:This results of the present study suggest that Fag t 2 promoter has potential as a seed-specific promoter, which can be used to activate the expression of functional genes at specific sites when aiming to improve crop yield and quality. The regulatory function of Fag t 2 promoter in plant stress resistance mediated by Fag t 2 should be explored in greater depth. © 2025 Society of Chemical Industry.

A novel super compact mutant, scp-3, was identified using map-based cloning in cucumber. The CsDWF7 gene encoding a delta7 sterol C-5(6) desaturase was the candidate gene of scp-3. Mining dwarf genes is important in understanding stem growth in crops. However, only a small number of dwarf genes have been cloned or characterized. Here, we characterized a cucumber (Cucumis sativus L.) dwarf mutant, super compact 3 (scp-3), which displays shortened internodes and dark green leaves with a wrinkled appearance. The photosynthetic rate of scp-3 is significantly lower than that of the wild type. The dwarf phenotype of scp-3 mutant can be partially rescued by the exogenous brassinolide (BL) application, and the endogenous brassinosteroids (BRs) levels in the scp-3 mutant were significantly lower compared to the wild type. Microscopic examination revealed that the reduced internode length in scp-3 resulted from a decrease in cell size. Genetic analysis showed that the dwarf phenotype of scp-3 was controlled by a single recessive gene. Combined with bulked segregant analysis and map-based cloning strategy, we delimited scp-3 locus into an 82.5 kb region harboring five putative genes, but only one non-synonymous mutation (A to T) was discovered between the mutant and its wild type in this region. This mutation occurred within the second exon of the CsGy4G017510 gene, leading to an amino acid alteration from Leu156 to His156. This gene encodes the CsDWF7 protein, an analog of the Arabidopsis DWF7 protein, which is known to be involved in the biosynthesis of BRs. The CsDWF7 protein was targeted to the cell membrane. In comparison to the wild type, scp-3 exhibited reduced CsDWF7 expression in different tissues. These findings imply that CsDWF7 is essential for both BR biosynthesis as well as growth and development of cucumber plants.

Parasitic weeds, such as Orobanche and Striga, threaten crops globally. Contiguous efforts on the discovery and development of structurally novel seed germination stimulants targeting HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) have been made with the goal of weed control. Here, we demonstrate that a natural compound dehydrocostus lactone (DCL) exhibits effective "suicide germination" activity against Orobanche cumana and covalently binds to OcKAI2d2 on two catalytic serine sites with the second modification dependent on the first one. The same interactions and covalent modifications of DCL are also confirmed in AtKAI2. Further in-depth evolution analysis indicates that the proposed two catalytic sites are present throughout the streptophyte algae, hornworts, lycophytes, and seed plants. This discovery is particularly noteworthy as it signifies the first confirmation of a plant endogenous molecule directly binding to KAI2, which is valuable for unraveling the elusive identity of the KAI2 ligand and for targeting KAI2 paralogues for the development of novel germination stimulants.

Hyaluronic acid (HA) is an acidic mucopolysaccharide of animal origin composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. Due to its excellent biocompatibility, biodegradability, and selective affinity for CD44 receptors on cell surfaces, HA is widely employed as a drug carrier. In our study, we aimed to target subcellular bacteria by grafting cystamine onto HA scaffolds through an amide reaction, producing a linker responsive to H2S and pH changes. Subsequently, hydrophobic dodecylamine was attached to HA, forming amphiphilic molecules. These amphiphilic entities can self-assemble into nanomicelles in an aqueous solution, thereby encapsulating the antibacterial agent triclosan (TCS). The resulting HA-based system (HASS-TCS) can be internalized via CD44-mediated endocytosis, releasing substantial amounts of streptomycin and TCS in H2S-rich and acidic environments. Additionally, HASS-TCS has demonstrated effectiveness in eradicating biofilms and addressing intracellular infections caused by Salmonella. This study underscores a novel pH-sensitive hyaluronic acid-based drug delivery system with significant potential for the effective treatment of intracellular infections.

Baculoviruses, the largest studied insect viruses, are highly pathogenic to host insects. Bombyx mori nucleopolyhedrovirus (BmNPV) is the main cause of nuclear polyhedrosis of silkworm, a viral disease that causes significant economic losses to the sericulture industry. The anti-BmNPV mechanism of the silkworm has not yet been characterized. Carboxypeptidase is an enzyme that is involved in virtually all life activities of animals and plants. Studies have shown that the carboxypeptidase family is related to insect immunity. There are few reports on the role of carboxypeptidase in the defense of silkworms against pathogen invasion. In this study, we identified the homologous gene Bombyx mori metal carboxypeptidases12 (BmMCP12) related to mammalian carboxypeptidase A2 (CPA2) and found that BmMCP12 had a Zn-pept domain. The BmMCP12 gene was primarily located in the cytoplasm and was highly expressed in the midgut of silkworms, and the expression level in BmN-SWU1 cells was upregulated after infection with BmNPV. After overexpression of the BmMCP12 gene, quantitative real-time (qRT)-PCR and Western blots showed that BmMCP12 could inhibit BmNPV replication, whereas knockout of the gene had the opposite effect. In addition, we constructed transgenic silkworm strains with a knockout of BmMCP12, and the transgenic strains had reduced resistance to BmNPV. These findings deepen the functional study of silkworm carboxypeptidase and provide a new target for BmNPV disease prevention in silkworms.

The fruit shape of cucumber is an important agronomic trait, and mining regulatory genes, especially dominant ones, is vital for cucumber breeding. In this study, we identified a short and fat fruit mutant, named sff, from an EMS mutagenized population. Compared to the CCMC (WT), sff (MT) exhibited reduced fruit length and increased dimeter. Segregation analysis revealed that the sff phenotype is controlled by a semi-dominant single gene with dosage effects. Through map-based cloning, the SFF locus was narrowed down to a 52.6 kb interval with two SNPs (G651A and C1072T) in the second and third exons of CsaV3_1G039870, which encodes an IQD family protein, CsSUN. The G651A within the IQ domain of CsSUN was identified as the unique SNP among 114 cucumber accessions, and it was the primary cause of the functional alteration in CsSUN. By generating CsSUN knockout lines in cucumber, we confirmed that CsSUN was responsible for sff mutant phenotype. The CsSUN is localized to the plasma membrane. CsSUN exhibited the highest expression in the fruit with lower expression in sff compared to WT. Histological observations suggest that the sff mutant phenotype is due to increased transverse cell division and inhibited longitudinal cell division. Transcriptome analysis revealed that CsSUN significantly affected the expression of genes related to cell division, expansion, and auxin signal transduction. This study unveils CsSUN's crucial role in shaping cucumber fruit and offers novel insights for cucumber breeding.

Mutations in the CsEMS1 gene result in male sterility and reduced wart number and density. Male sterility and fruit wart formation are two significant agronomic characteristics in cucumber (Cucumis sativus), yet knowledge of our underlying genetics is limited. In this study, we identified an EMS-induced male sterility and few small warts mutant (msfsw). Histological observations revealed defects the absence of tapetum, meiotic aberration and impaired microspore formation in the anthers of the mutant. The mutant also exhibits a reduction in both the size and number of fruit spines and fruit tubercules. Genetic analysis revealed that a single recessive gene is responsible for the mutant phenotypes. BSA-Seq and fine genetic mapping mapped the msfsw locus to a 63.7 kb region with four predicted genes. Multiple lines of evidence support CsEMS1(CsaV3_3G016940) as the candidate for the mutant allele which encodes an LRR receptor-like kinase, and a non-synonymous SNP inside the exon of CsEMS1 is the causal polymorphisms for the mutant phenotypes. This function of CsEMS1 in determination of pollen fertility was confirmed with generation and characterization of multiple knockout mutations with CRISPR/Cas9 based gene editing. In the wild-type (WT) plants, CsEMS1 was highly expressed in male flowers. In the mutant, the expression level of CsEMS1, several tapetum identity-related genes, and trichome-related genes were all significantly reduced as compared with the wild-type. Protein–protein interaction assays revealed physical interactions between CsEMS1 and CsTPD1. Quantitation of endogenous phytohormones revealed a reduction in the ethylene precursor ACC in CsEMS1 knockout lines. This work identified an important role of CsEMS1 in anther and pollen development as well as fruit spine/wart development in cucumber.

Map-based cloning revealed that a mutation in a highly conserved amino acid of the CsGME gene encoding GDP-mannose 3,5-epimerase, causes the phenotype of little and wrinkled leaves in cucumbers. Leaf size is a critical determinant of plant architecture in cucumbers, yet only a few genes associated with this trait have been mapped or cloned. Here, we identified and characterized a mutant with little and wrinkled leaves, named lwl-1. Genetic analysis revealed that the phenotype of the lwl-1 was controlled by a single recessive gene. Through map-based cloning, the lwl-1 locus was narrowed down to a 12.22-kb region exclusively containing one fully annotated gene CsGME (CsaV3_2G004170). CsGME encodes GDP-mannose 3,5-epimerase, which is involved in the synthesis of ascorbic acid (ASA) and one of the components of pectin, RG-II. Whole-length sequencing of the 12.22 kb DNA fragment revealed the presence of only a non-synonymous mutation located in the sixth exon of CsGME in lwl-1, resulting in an amino acid alteration from Pro363 to Leu363. This mutation was unique among 118 inbred lines from cucumber natural populations. CsGME expression significantly reduced in various organs of lwl-1, accompanied by a significant decrease in ASA and pectin content in leaves. Both CsGME and Csgme proteins were localized to the cytoplasm. The mutant phenotype exhibited partial recovery after the application of exogenous boric acid. Silencing CsGME in cucumber through VIGS confirmed its role as the causal gene for lwl-1. Transcriptome profiling revealed that CsGME greatly affected the expression of genes related to the cell division process and cell plate formation. This study represents the first report to characterize and clone the CsGME in cucumber, indicating its crucial role in regulating leaf size and development.

The novel spontaneous long hypocotyl and early flowering (lhef) mutation in cucumber is due to a 5551-bp LTR-retrotransposon insertion in CsPHYB gene encoding PHYTOCHROME B, which plays a major role in regulating photomorphogenic hypocotyl growth and flowering. Hypocotyl length and flowering time are important for establishing high-quality seedlings in modern cucumber production, but little is known for the underlying molecular mechanisms of these two traits. In this study, a spontaneous cucumber long hypocotyl and early flowering mutant was identified and characterized. Based on multiple lines of evidence, we show that cucumber phytochrome B (CsPHYB) is the candidate gene for this mutation, and a 5551-bp LTR-retrotransposon insertion in the first exon of CsPHYB was responsible for the mutant phenotypes. Uniqueness of the mutant allele at CsPHYB was verified in 114 natural cucumber lines. Ectopic expression of the CsPHYB in Arabidopsis phyB mutant rescued the long hypocotyl and early flowering phenotype of phyB-9 mutant. The wild-type CsPHYB protein was localized on the membrane and cytoplasm under white light condition, whereas in the nucleus under red light, it is consistent with its roles as a red-light photoreceptor in Arabidopsis. However, the mutant csphyb protein was localized on the membrane and cytoplasm under both white and red-light conditions. Expression dynamics of CsPHYB and several cell elongation-related genes were positively correlated with hypocotyl elongation; the transcription levels of key positive and negative regulators for flowering time were also consistent with the anthesis dates in the mutant and wild-type plants. Yeast two hybrid and bimolecular fluorescence complementation assays identified physical interactions between CsPHYB and phytochrome interacting factor 3/4 (CsPIF3/4). These findings will provide new insights into the roles of the CsPHYB in cucumber hypocotyl growth and flowering time.

The compact (cp) phenotype in cucumber (Cucumis sativus L.) is an important plant architecture-related trait with a great potential for cucumber improvement. In this study, we conducted map-based cloning of the cp locus, identified and functionally characterized the candidate gene. Comparative microscopic analysis suggested that the short internode in the cp mutant is due to fewer cell numbers. Fine genetic mapping delimited cp into an 8.8-kb region on chromosome 4 harboring only one gene, CsERECTA (CsER) that encodes a leucine-rich repeat receptor-like kinase. A 5.5-kb insertion of a long terminal repeat retrotransposon in the 22nd exon resulted in loss-of-function of CsER in the cp plant. Spatiotemporal expression analysis in cucumber and CsER promoter-driven GUS assays in Arabidopsis indicated that CsER was highly expressed in the stem apical meristem and young organs, but the expression level was similar in the wild type and mutant cucumber plants. However, CsER protein accumulation was reduced in the mutant as revealed by western hybridization. The mutation in cp also did not seem to affect self-association of CsER for formation of dimers. Ectopic expression of CsER in Arabidopsis was able to rescue the plant height of the loss-of-function AtERECTA mutant, whereas the compact inflorescence and small rosette leaves of the mutant could be partially recovered. Transcriptome profiling in the mutant and wild type cucumber plants revealed hormone biosynthesis/signaling, and photosynthesis pathways associated with CsER-dependent regulatory network. Our work provides new insights for the use of cp in cucumber breeding.

LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP), a neuroactive metabolite distributed widely in several plant species including Panax notoginseng, Panax ginseng, and L. sativus. The enzymatic activity of LsSAT2 is post-translationally regulated by its involvement in the cysteine regulatory complex in mitochondria via interaction with β-CAS (β-cyanoalanine synthase). In this study, the binding sites of LsSAT2 with the substrate Ser were first determined as Glu290, Arg316, and His317 and the catalytic sites were determined as Asp267, Asp281, and His282 via site-directed/truncated mutagenesis, in vitro enzymatic activity assay, and functional complementation of the SAT-deficient Escherichia coli strain JM39. Furthermore, the C-terminal 10-residue peptide of LsSAT2 is confirmed to be critical to interact with LsCAS, and Ile336 in C10 peptide is the critical amino acid. These results will enhance our understanding of the regulation of LsSAT2 activities and the biosynthesis of β-ODAP in L. sativus.

Pathogenic bacteria residing inside cells could cause disruption of cellular metabolic balance. Therefore, basing on high oxidative stress response of the intracellular bacteria infected micro-environment, a novel amphipathic micelle (HATAD-TCS) was developed consisting of hyaluronic acid-derivative and reactive oxygen species (ROS) - responsive group and antibacterial agent triclosan (TCS). ROS-generating cinnamaldehyde (CA) was incorporated into ROS-cleavable linkages which are future linked to the 1-decylamine to form hydrophobicity. The cinnamaldehyde released did not just killed bacteria however, also maintained intracellular ROS levels. In this study, the HATAD-TCS micelles have been characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The HATAD-TCS micelles could release drug gradually upon exposure to endogenous ROS being caused by infected intracellular bacteria. Furthermore, the more promising therapeutic effect of the HATAD-TCS micelles was observed in a mouse pneumonia model. These results might highlight a ROS-responsive hyaluronic acid-based nanoparticle, which could effectively treat intracellular bacterial infections.

Grass pea (Lathyrus sativus L.) is a robust legume crop with high protein content and good stress tolerance. However, it needs genetic improvement due to the presence of several anti-nutritional factors like the neurotoxic β-N-oxalyl-L-α, β-diaminopropionic acid (β-ODAP), etc. To investigate β-ODAP metabolism, quantitative real-time PCR would be a strong tool in gene expression analysis. Therefore, selection of reference genes in grass pea is necessary to get reliable and accurate results. In this study, eleven candidate reference genes, including 60SRP, ABCT, PP2C, IDE3, EF1α, ElF1b, Fbox, GAPDH, HSP80, IF4α and pUBQ were selected for identifying expression stability in different tissues under different developmental stages or drought stress. Expression stability was calculated using geNorm, NormFinder, BestKeeper separately, compared and ranked via RefFinder. The optimal number of reference genes required for normalization was determined by pairwise variation (Vn/Vn + 1) using geNorm. EF1α and Elf1b were recommended as the most stable reference genes in drought stress tissues, whereas IF4α, HSP80 and Elf1b exhibited the highest stability in developmental stage tissues and Elf1b, IF4α and ABCT in total samples set. Moreover, two or more reference genes were recommended for qRT-PCR analysis under our experimental conditions. As the first report of reference gene evaluation in grass pea, our study will facilitate further studies in gene functions related to β-ODAP metabolism and grass pea improvement.