Protein research in millets: current status and way forward.

MAIN CONCLUSION: Millets' protein studies are lagging behind those of major cereals. Current status and future insights into the investigation of millet proteins are discussed. Millets are important small-seeded cereals majorly grown and consumed by people in Asia and Africa and are considered crops of future food security. Although millets possess excellent climate resilience and nutrient supplementation properties, their research advancements have been lagging behind major cereals. Although considerable genomic resources have been developed in recent years, research on millet proteins and proteomes is currently limited, highlighting a need for further investigation in this area. This review provides the current status of protein research in millets and provides insights to understand protein responses for climate resilience and nutrient supplementation in millets. The reference proteome data is available for sorghum, foxtail millet, and proso millet to date; other millets, such as pearl millet, finger millet, barnyard millet, kodo millet, tef, and browntop millet, do not have any reference proteome data. Many studies were reported on stress-responsive protein identification in foxtail millet, with most studies on the identification of proteins under drought-stress conditions. Pearl millet has a few reports on protein identification under drought and saline stress. Finger millet is the only other millet to have a report on stress-responsive (drought) protein identification in the leaf. For protein localization studies, foxtail millet has a few reports. Sorghum has the highest number of 40 experimentally proven crystal structures, and other millets have fewer or no experimentally proven structures. Further proteomics studies will help dissect the specific proteins involved in climate resilience and nutrient supplementation and aid in breeding better crops to conserve food security.

Long-term fertilization and manuring effects on the nexus between sulphur distribution and SOC in an Inceptisol over five decades under a finger millet-maize cropping system.

Our investigation revealed that alterations in sulphur (S) pools are predominantly governed by soil organic carbon (SOC), soil nitrogen (N), microbial biomass, and soil enzyme activities in sandy clay loam (Vertic Ustropept) soil. We employed ten sets of nutrient management techniques, ranging from suboptimal (50% RDF) to super-optimal doses (150% RDF), including NPK + Zn, NP, N alone, S-free NPK fertilizers, NPK + FYM, and control treatments, to examine the interrelation of S with SOC characteristics. Fourier-transform infrared (FT-IR) spectroscopy was utilized to analyze the functional groups present in SOC characterization across four treatments: 100% NPK, 150% NPK, NPK + FYM, and absolute control plots. Principal component analysis (PCA) was then applied to assess 29 minimal datasets, aiming to pinpoint specific soil characteristics influencing S transformation. In an Inceptisol, the application of fertilizers (100% RDF) in conjunction with 10 t ha-1 of FYM resulted in an increase of S pools from the surface to the subsurface stratum (OS > HSS > SO42--S > WSS), along with an increase in soil N and SOC. FT-IR spectroscopy identified cellulose and thiocyanate functional groups in all four plots, with a pronounced presence of carbohydrate-protein polyphenol, sulfoxide (S=O), and nitrate groups specifically observed in the INM plot. The PCA findings indicated that the primary factors influencing soil quality and crop productivity (r2 of 0.69) are SOC, SMBC, SMBN, SMBS, and the enzyme activity of URE, DHA, and AS. According to the study, the combined application of fertilizer and FYM (10 t ha-1) together exert a positive impact on sulphur transformation, SOC accumulation, and maize yield in sandy clay loam soil.

Fermentation of millet bran with Bacillus natto: enhancement of bioactivity levels and the bioactivity of bran extract.

BACKGROUND: Millet bran (MB), a byproduct of millet production, is rich in functional components but it is underutilized. In recent years, researchers have shown that fermentation can improve the biological activity of cereals and their byproducts. This study used Bacillus natto to ferment millet bran to improve its added value and broaden the application of MB. The bioactive component content, physicochemical properties, and functional activity of millet bran extract (MBE) from fermented millet bran were determined. RESULTS: After fermentation, the soluble dietary fiber (SDF) content increased by 92.0%, the ß-glucan content by 164.4%, the polypeptide content by 111.4%, the polyphenol content by 32.5%, the flavone content by 16.4%, and the total amino acid content by 95.4%. Scanning electron microscopy revealed that the microscopic morphology of MBE changed from complete and dense blocks to loosely porous shapes after fermentation. After fermentation, the solubility, water-holding capacity, and viscosity significantly increased and the particle size decreased. Moreover, the glucose adsorption capacity (2.1 mmol g-1), glucose dialysis retardation index (75.3%), and α-glucosidase inhibitory (71.4%, mixed reversible inhibition) activity of the fermented MBE (FMBE) were greater than those of the unfermented MBE (0.99 mmol g-1, 32.1%, and 35.1%, respectively). The FMBE presented better cholesterol and sodium cholate (SC) adsorption properties and the adsorption was considered inhomogeneous surface adsorption. CONCLUSION: Fermentation increased the bioactive component content and improved the physicochemical properties of MBE, thereby improving its hypoglycemic and hypolipidemic properties. This study not only resolves the problem of millet bran waste but also encourages the development of higher value-added application methods for millet bran. © 2024 Society of Chemical Industry.

Comprehensive physiological, transcriptomic, and metabolomic analysis of the key metabolic pathways in millet seedling adaptation to drought stress.

Drought is one of the leading environmental constraints that affect the growth and development of plants and, ultimately, their yield and quality. Foxtail millet (Setaria italica) is a natural stress-resistant plant and an ideal model for studying plant drought resistance. In this study, two varieties of foxtail millet with different levels of drought resistance were used as the experimental material. The soil weighing method was used to simulate drought stress, and the differences in growth, photosynthetic physiology, metabolite metabolism, and gene transcriptional expression under drought stress were compared and analyzed. We aimed to determine the physiological and key metabolic regulation pathways of the drought-tolerant millet in resistance to drought stress. The results showed that drought-tolerant millet exhibited relatively stable growth and photosynthetic parameters under drought stress while maintaining a relatively stable level of photosynthetic pigments. The metabolomic, transcriptomic, and gene co-expression network analysis confirmed that the key to adaptation to drought by millet was to enhance lignin metabolism, promote the metabolism of fatty acids to be transformed into cutin and wax, and improve ascorbic acid circulation. These findings provided new insights into the metabolic regulatory network of millet adaptation to drought stress.

Antioxidant Peptides Derived from Millet Bran Promote Longevity and Stress Resistance in Caenorhabditis elegans.

Millet bran as a by-product of millet grain processing remains a reservoir of active substances. In this study, functional millet bran peptides (MBPE) were obtained from bran proteins after alcalase hydrolysis and ultrafiltration. The activity of MBPE was assessed in vitro and in the model organism Caenorhabditis elegans (C. elegans). In vitro, compared to unhydrolyzed proteins, MBPE significantly enhanced the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate (ABTS) and hydroxyl radicals scavenging activity, and the scavenging rate of MBPE with 15,000 U/g alcalase reached 42.79 ± 0.31%, 61.38 ± 0.41 and 45.69 ± 0.84%, respectively. In C. elegans, MBPE at 12.5 µg/mL significantly prolonged the lifespan by reducing lipid oxidation, oxidative stress, and lipofuscin levels. Furthermore, MBPE increased the activities of the antioxidant enzymes. Genetic analyses showed that MBPE-mediated longevity was due to a significant increase in the expression of daf-16 and skn-1, which are also involved in xenobiotic and oxidative stress responses. In conclusion, this study found that MBPE had antioxidant and life-prolonging effects, which are important for the development and utilization of millet bran proteins as resources of active ingredients.

Compound probiotics producing cellulase could replace cellulase preparations during solid-state fermentation of millet bran.

Low-value agricultural by-products can be converted into high-value biological products by fermentation with probiotic strains or by enzymatic hydrolysis. However, the high costs of enzyme preparations significantly limit their applications in fermentation. In this study, the solid-state fermentation of millet bran was performed using a cellulase preparation and compound probiotics producing cellulase (CPPC), respectively. The results showed that both factors effectively destroyed the fiber structure, reduced the crude fiber content by 23.78% and 28.32%, respectively, and significantly increased the contents of beneficial metabolites and microorganisms. Moreover, CPPC could more effectively reduce the anti-nutrient factors and increase the content of anti-inflammatory metabolites. The correlation analysis revealed that Lactiplantibacillus and Issatchenkia had synergistic growth during fermentation. Overall, these results suggested that CPPC could replace cellulase preparation and improve antioxidant properties while reducing anti-nutrient factors of millet bran, thus providing a theoretical reference for the efficient utilization of agricultural by-products.

Microscopic assessment of the degradation of millet starch granules by endogenous and exogenous enzymes during mashing.

The high gelatinization temperature (GT) of millet starch prevents the usage of infusion or step mashes as an effective means to generate fermentable sugars (FS) in brewing because the malt amylases lack thermostability at GT. Here, we investigate processing modifications to determine if millet starch can be efficiently degraded below GT. We determined that producing finer grists through milling did not introduce enough granule damage to markedly change gelatinization characteristics, though there was improved liberation of the endogenous enzymes. Alternatively, exogenous enzyme preparations were added to investigate their ability to degrade intact granules. At the recommended dosages (0.625 µL/g malt), significant FS concentrations were observed, although at lower concentrations and with a much-altered profile than possible with a typical wort. When exogenous enzymes were introduced at high (10×) addition rates, significant losses of granule birefringence and granule hollowing were observed well below GT, suggesting these exogenous enzymes can be utilized to digest millet malt starch below GT. The exogenous maltogenic α-amylase appears to drive the loss of birefringence, but more research is needed to understand the observed predominate glucose production.

Millet Bran Protein Hydrolysate Displays the Anti-non-alcoholic Fatty Liver Disease Effect via Activating Peroxisome Proliferator-Activated Receptor γ to Restrain Fatty Acid Uptake.

Non-alcoholic fatty liver disease (NAFLD) is a serious health problem worldwide. Impeding fatty acid uptake may be an attractive therapeutic strategy for NAFLD. In the current study, we found that millet bran protein hydrolysate (MBPH) prepared by in vitro gastrointestinal bionic digestion exhibits the potential of anti-NAFLD in vitro and in vivo, characterized by the alleviation of hepatic steatosis and the reduction of lipid accumulation. Further, MBPH significantly decreased the expression levels of fatty acid uptake related genes (FABP1, FABP2, FABP4, CD36, and CPT-1α) of liver tissue in a NAFLD mice model through activating peroxisome proliferator-activated receptor γ (PPARγ) and efficiently restrained the fatty acid uptake of liver tissue, thus exerting anti-NAFLD activity. As expected, the anti-NAFLD effect induced by MBPH, characterized by the alleviation of hepatic vacuolar degeneration, hepatic steatosis, and fibrosis, was effectively abrogated with PPARγ inhibitor (GW9662) treatment. These results indicate that the retardant of fatty acid uptake induced by PPARγ activation may be the critical factor for the anti-NAFLD effect of MBPH. Collectively, MBPH has the potential as a next-generation dietary supplementation for the prevention and treatment of NAFLD.

Implication of solvent polarities on browntop millet (Urochloa ramosa) phenolic antioxidants and their ability to protect oxidative DNA damage and inhibit α-amylase and α-glucosidase enzymes.

Phenolics of browntop millet extracted in solvents with varying polarities [water, methanol, acetone (80%), ethanol (70%)] were comparatively assessed for their phenolic profiles, antioxidant activities, DNA damage protection and enzyme inhibitory properties. Results indicated that acetone (80%) and ethanol (70%) were most effective in extracting millet phenolics than other solvents. Gallic, caffeic and ferulic acids were the major phenolic acids, myricetin and kaempferol were the most abundant flavonoids detected in all the extracts of browntop millet. Phenolics extracted in 80% acetone and 70% ethanol offered noticeable contributions toward several antioxidant mechanisms and prevented the oxidative DNA damage than water and methanol extracts. All the millet extracts exhibited potent inhibition towards α-glucosidase than α-amylase activities. These results suggest that the solvents and their polarities impacted the extraction and bioactivities of millet phenolics and provided useful information for the effective utilization of browntop millet as a functional food ingredient to manage hyperglycemia.

Millet-inspired systems metabolic engineering of NUE in crops.

The use of nitrogen (N) fertilizers in agriculture has a great ability to increase crop productivity. However, their excessive use has detrimental effects on the environment. Therefore, it is necessary to develop crop varieties with improved nitrogen use efficiency (NUE) that require less N but have substantial yields. Orphan crops such as millets are cultivated in limited regions and are well adapted to lower input conditions. Therefore, they serve as a rich source of beneficial traits that can be transferred into major crops to improve their NUE. This review highlights the tremendous potential of systems biology to unravel the enzymes and pathways involved in the N metabolism of millets, which can open new possibilities to generate transgenic crops with improved NUE.

5M approach to decipher starch-lipid interaction in minor millets.

KEY MESSAGE: The 5M approach can be applied to understand genetic complexity underlying nutritional traits of minor millets. It will help to systematically identify genomic regions/candidate genes imprinting metabolite profiles.

Preparation and characterization of a novel nanoemulsion consisting of chitosan and Cinnamomum tamala essential oil and its effect on shelf-life lengthening of stored millets.

This study aimed to improve the stability of Cinnamomum tamala essential oil (CTEO) via encapsulating into chitosan nanoemulsion (CsNe) through an ionic-gelation technique and explore its food preservative efficacy against aflatoxigenic strain of Aspergillus flavus (AFLHPSi-1, isolated from stored millet), aflatoxin B1 (AFB1) contamination, and lipid peroxidation, causing qualitative deterioration of stored millets. The CTEO was characterized through gas chromatography-mass spectrometry (GC-MS) analysis that confirmed the presence of linalool as a major component occupying approximately 82.64% of the total oil. The synthesized nanoparticles were characterized through scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis. The encapsulation efficiency (EE) and loading capacity (LC) of CTEO-CsNe were found to be 97.71% and 3.33%, respectively. In vitro release study showed a biphasic release pattern: with an initial burst release followed by a controlled release of CTEO. During investigation of efficacy, the CTEO-CsNe caused complete inhibition of A. flavus growth, and AFB1 biosynthesis at 1.0 and 0.8 µL/mL, respectively. The CTEO-CsNe exhibited its antifungal mode of action by altering fungal plasma membrane integrity (ergosterol inhibition) and permeability (leakage of important cellular constituents), and antiaflatoxigenic mode of action by inhibiting cellular methylglyoxal biosynthesis. CTEO-CsNe showed high free radical scavenging capacity (IC50 = 5.08 and 2.56 µL/mL) against DPPH•+ and ABTS•+ radicals, respectively. In addition, CTEO-CsNe presented remarkable preservative efficacy, inhibiting AFB1 and lipid peroxidation in model food system (Setaria italica) without altering their organoleptic properties. Based on overall results, CTEO-CsNe can be recommended as a novel shelf-life enhancer of stored millet samples.

Transcriptome Sequencing and Metabolome Analysis Reveals the Molecular Mechanism of Drought Stress in Millet.

As one of the oldest agricultural crops in China, millet (Panicum miliaceum) has powerful drought tolerance. In this study, transcriptome and metabolome analyses of 'Hequ Red millet' (HQ) and 'Yanshu No.10' (YS10) millet after 6 h of drought stress were performed. Transcriptome characteristics of drought stress in HQ and YS10 were characterized by Pacbio full-length transcriptome sequencing. The pathway analysis of the differentially expressed genes (DEGs) showed that the highly enriched categories were related to starch and sucrose metabolism, pyruvate metabolism, metabolic pathways, and the biosynthesis of secondary metabolites when the two millet varieties were subjected to drought stress. Under drought stress, 245 genes related to energy metabolism were found to show significant changes between the two strains. Further analysis showed that 219 genes related to plant hormone signal transduction also participated in the drought response. In addition, numerous genes involved in anthocyanin metabolism and photosynthesis were confirmed to be related to drought stress, and these genes showed significant differential expression and played an important role in anthocyanin metabolism and photosynthesis. Moreover, we identified 496 transcription factors related to drought stress, which came from 10 different transcription factor families, such as bHLH, C3H, MYB, and WRKY. Further analysis showed that many key genes related to energy metabolism, such as citrate synthase, isocitrate dehydrogenase, and ATP synthase, showed significant upregulation, and most of the structural genes involved in anthocyanin biosynthesis also showed significant upregulation in both strains. Most genes related to plant hormone signal transduction showed upregulated expression, while many JA and SA signaling pathway-related genes were downregulated. Metabolome analysis was performed on 'Hequ red millet' (HQ) and 'Yanshu 10' (YS10), a total of 2082 differential metabolites (DEMs) were identified. These findings indicate that energy metabolism, anthocyanins, photosynthesis, and plant hormones are closely related to the drought resistance of millet and adapt to adversity by precisely regulating the levels of various molecular pathways.

The influences of acetylation, hydroxypropylation, enzymatic hydrolysis and crosslinking on improved adsorption capacities and in vitro hypoglycemic properties of millet bran dietary fibre.

The effects of acetylation, hydroxypropylation, cellulase hydrolysis and crosslinking on adsorption capacities and in vitro hypoglycemic activities of millet bran dietary fibre (MBDF) were studied. The results demonstrated that both acetylation and hydroxypropylation improved water swelling ability of MBDF, and adsorption capacities of cholesterol, cholate and copper ion on MBDF. Acetylation and hydroxypropylation also enhanced α-glucosidase and α-amylase inhibition activities, glucose-binding ability and glucose diffusion retardation index (GDRI) of MBDF. Acetylated MBDF showed the highest cholate (77.31 mg/g) and cholesterol (13.97 mg/g) adsorption capacities. The crosslinking improved adsorption of cholate, cholesterol, copper ion (25.64 mg/g) and nitrite ion (181.59 µg/g) on MBDF; but reduced α-amylase inhibition activity (p < 0.05). Moreover, cellulase hydrolyzed MBDF exhibited the highest GDRI (39.60%) and α-amylase inhibition activity (34.53%), but the lowest oil and cholate adsorption capacities. The results suggest that the modified MBDFs can be used as an ingredient of hypoglycemic foods.

Millet shell polyphenols prevent atherosclerosis by protecting the gut barrier and remodeling the gut microbiota in ApoE-/- mice.

Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease. The anti-inflammatory effect of certain polyphenols has been recognized. Active polyphenols were extracted from millet shells (MSPs), and their main components including 3-hydroxybenzylhydrazine, luteolin-3',7-diglucoside, N-acetyltyramine, p-coumaric acid, vanillin, sinapic acid, ferulic acid and isophorone exhibited the anti-atherosclerotic potential in vitro. To explore the anti-atherosclerotic activity of MSPs in vivo, a classic atherosclerosis model was constructed in ApoE-/- mice fed with a high-fat diet. The results showed that MSPs effectively inhibited the development of atherosclerotic plaques in the aorta and reduced the levels of lipopolysaccharide (LPS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). A further study found that the expression of tight junction proteins (occludin, zona occludens-1 and claudin1) was obviously up-regulated in the MSPs-treated group at the mRNA and protein levels. Interestingly, MSPs significantly changed the structure of gut microbiota in ApoE-/- mice with a high-fat diet, which is characterized by the enriched Oscillospira and Ruminococcus, and the abridged Allobaculum at the genus level. Collectively, these results suggest that MSPs regulate the integrity of the gut barrier and the structure of the gut microbiota, ultimately inhibiting the development of atherosclerotic plaques. This study provides new insights into the potential cardiovascular protective effects induced by millet shell polyphenols.

Improvement of nutrient bioavailability in millets: Emphasis on the application of enzymes.

Millets are a traditional staple food of the dryland regions of the world and are rich in essential nutrients like protein, fatty acids, minerals, vitamins, and dietary fiber. Also, millets commonly synthesize a range of secondary metabolites to protect themselves against adverse conditions. These factors are collectively termed anti-nutritional factors and the existence of these factors in millets might reduce the accessibility of the nutrients in humans. Some of these factors include protease inhibitors, tannins, non-starch polysaccharides-glucans, phytates, and oxalates each of which might directly or indirectly affect the digestibility of nutrients. Methods like soaking, germination, autoclaving, debranning, and the addition of exogenous enzymes have been used to reduce the anti-nutritional factors and elevate the bioavailability of the nutrients. This review summarizes various methods that have been used to improve nutrient bioavailability, specifically emphasizing the use of enzymes to improve nutrient bioavailability from millets. © 2021 Society of Chemical Industry.

Maize ANT1 modulates vascular development, chloroplast development, photosynthesis, and plant growth.

Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.

Humic acid improves the physiological and photosynthetic characteristics of millet seedlings under drought stress.

We aimed to determine whether humic acid (HA) can alleviate the injury of millet caused by drought and its potential mechanism. Millet seeds (Jingu 21 and Zhangza 10) were soaked in different concentrations of HA (0, 50, 10, 200, and 300 mg L-1) for 12 h. The physiological and photosynthetic characteristics of millet seedlings, including growth parameters, osmotic regulators, antioxidase activity, photosynthesis, chlorophyll fluorescence, and P700 parameters, were determined before and after drought stress. HA significantly promoted the growth of millet seedlings under drought stress. Pretreatment with 100 mg L-1 or 200 mg L-1 HA significantly increased free proline, soluble protein, and activity of the antioxidant enzyme system (superoxide dismutase, peroxidase, and catalase) in both Zhangza 10 and Jingu 21. The accumulation of reactive oxygen species ([Formula: see text] and H2O2) was reduced in HA treatments compared with that of the control (P < .05). Moreover, HA (100 mg L-1) significantly increased net photosynthetic rate, stomatal conductance, effective quantum yield of photosystem II, relative photosynthetic electron transfer rate of photosystem II, and photochemical quenching. HA also reduced intercellular CO2 concentration and non-photochemical quenching. Furthermore, 200 mg L-1 HA significantly increased the maximum P700, effective quantum yield of photosystem I, and relative photosynthetic electron transfer rate of photosystem I in Zhangza 10 and decreased non-photochemical energy dissipation in Jingu 21 and Zhangza 10 under drought stress. HA promoted the growth of millet seedlings under drought stress by promoting the osmotic adjustment ability and antioxidant capacity of seedlings and increased photosynthesis.

Changes in protein structural characteristics upon processing of gluten-free millet pasta.

Proso millet exhibits favorable agronomic and nutritional properties but is currently under-utilized in the northern hemisphere. This study compared processing-induced changes in protein characteristics of commercial pasta to fresh gluten-free pasta from proso millet varieties differing in prolamin profile. Protein solubility, accessible thiols and secondary structures were measured in dough, sheeted and cooked pasta. Relationships between protein conformation and characteristics related to pasta quality were determined. Cooking significantly lowered protein solubility and induced exposure of thiol groups as well as a shift in secondary structure distribution, while sheeting only had a minor effect. Random structures positively and significantly (P < 0.05) correlated with solubility, cooking loss and protein digestibility. In contrast, ß-sheets, the main secondary structure in cooked pasta, negatively correlated with these properties. The utilization of proso millet in gluten-free pasta is promising, however, processing optimization to elicit targeted protein modifications to balance quality and nutritional attributes requires further investigation.

Deep-rooted pigeon pea promotes the water relations and survival of shallow-rooted finger millet during drought-Despite strong competitive interactions at ambient water availability.

Bioirrigation has been defined as the transfer of hydraulically lifted water by a deep-rooted plant to a neighbouring shallow-rooted plant which cannot access deep soil moisture. In this study, we tested if facilitative effects of bioirrigation or the competition for water dominate the interaction of two intercropped plants-deep-rooted pigeon pea (PP) and shallow-rooted finger millet (FM) before and during a drought. Additionally, we tested how the presence of a common mycorrhizal network (CMN) affects the balance between facilitative (i.e. bioirrigation) and competitive interactions between two intercropping species. Our results show that PP can indeed promote the water relations of FM during a drought event. Specifically, stomatal conductance in FM controls dropped to low values of 27.1 to 33.6 mmol m-2s-1, while FM in intercropping treatments were able to maintain its stomatal conductance at 60 mmol m-2s-1. In addition, the presence of PP reduced the drought-induced foliar damage and mortality of FM. The observed facilitative effects of PP on FM were partially enhanced by the presence of a CMN. In contrast to the facilitative effects under drought, PP exerted strong competitive effects on FM before the onset of drought. This hindered growth and biomass production of FM when intercropped with PP, an effect that was even enhanced in the presence of a CMN. The results from our study thus indicate that in intercropping, deep-rooted plants may act as "bioirrigators" for shallow-rooted crops and that a CMN can promote these facilitative effects. However, the interspecific competition between the intercropped plants under conditions of abundant moisture supply can be strong and are enhanced by the presence of a CMN. In more general terms, our study shows that the extent by which the antagonistic effects of facilitation and competition are expressed in an intercropping system strongly depends on the availability of resources, which in the case of the present study was water and the presence of biotic interactions (i.e. the presence of a CMN).