NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

LC-MS and MALDI-MSI-based metabolomic approaches provide insights into the spatial-temporal metabolite profiles of Tartary buckwheat achene development.

Tartary buckwheat, celebrated as the "king of grains" for its flavonoid and phenolic acid richness, has health-promoting properties. Despite significant morphological and metabolic variations in mature achenes, research on their developmental process is limited. Utilizing Liquid chromatography-mass spectrometry and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging, we conducted spatial-temporal metabolomics on two cultivars during achene development. Metabolic profiles including 17 phenolic acids and 83 flavonoids are influenced by both varietal distinctions and developmental intricacies. Notably, flavonols, as major flavonoids, accumulated with achene ripening and showed a tissue-specific distribution. Specifically, flavonol glycosides and aglycones concentrated in the embryo, while methylated flavonols and procyanidins in the hull. Black achenes at the green achene stage have higher bioactive compounds and enhanced antioxidant capacity. These findings provide insights into spatial and temporal characteristics of metabolites in Tartary buckwheat achenes and serve as a theoretical guide for selecting optimal resources for food production.

Plant anthraquinones: Classification, distribution, biosynthesis, and regulation.

Anthraquinones are polycyclic compounds with an unsaturated diketone structure (quinoid moiety). As important secondary metabolites of plants, anthraquinones play an important role in the response of many biological processes and environmental factors. Anthraquinones are common in the human diet and have a variety of biological activities including anticancer, antibacterial, and antioxidant activities that reduce disease risk. The biological activity of anthraquinones depends on the substitution pattern of their hydroxyl groups on the anthraquinone ring structure. However, there is still a lack of systematic summary on the distribution, classification, and biosynthesis of plant anthraquinones. Therefore, this paper systematically reviews the research progress of the distribution, classification, biosynthesis, and regulation of plant anthraquinones. Additionally, we discuss future opportunities in anthraquinone research, including biotechnology, therapeutic products, and dietary anthraquinones.

Visualization and identification of benzylisoquinoline alkaloids in various nelumbo nucifera tissues.

Benzylisoquinoline alkaloids in lotus (Nelumbo nucifera) seed plumules and leaves exhibit significant tissue specificity for their pharmacological effects and potential nutritional properties. Herein, 46 benzylisoquinoline alkaloids were identified via UPLC-QTOF-HRMS, of which 9 were annotated as glycosylated monobenzylisoquinoline alkaloids concentrated in the seed plumules. The spatial distribution of targeted benzylisoquinoline alkaloids in leaves, seed plumules, and milky sap was determined via MALDI-MSI. Furthermore, 37 Nelumbo cultivars were investigated using targeted metabolomics to provide insights into functional tea development. While aporphine alkaloids comprised the main compounds present in lotus leaves, bisbenzylisoquinoline alkaloids were the main compounds in lotus plumules, where glycosylation primarily occurs. These findings can help understand the distribution of benzylisoquinoline alkaloids in lotus tissue and the directional breeding of varieties enriched with specific chemical functional groups for nutritional and pharmacological applications.

Functional characterization and key residues engineering of a regiopromiscuity O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis in Nelumbo nucifera.

Lotus (Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study we identified the regiopromiscuity of an O-methyltransferase, designated NnOMT6, isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O, aporphine skeleton 6-O, phenylpropanoid 3-O, and protoberberine 2-O. We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that residues D316, N130, L135, N176A, D269, and E328 were critical for BIA O-methyltransferase activities; furthermore, N323A, a mutant of NnOMT6, demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6. To the best of our knowledge, this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N. nucifera. The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.

Comprehensive genome-wide identification and functional characterization of MAPK cascade gene families in Nelumbo.

Mitogen-activated protein kinase (MAPK) cascade signaling pathway plays pivotal roles in various plant biological processes. However, systematic study of MAPK cascade gene families is yet to be conducted in lotus. Herein, 198 putative MAPK genes, including 152 MAP3Ks, 15 MKKs, and 31 MPKs genes were identified in Nelumbo. Segmental duplication was identified as the predominant factor driving MAPK cascade gene family expansion in lotus. MAPK cascade genes in N. nucifera and N. lutea shared high degree of sequence homologies, with 84, 9, and 19 homologous MAP3K, MKK, and MPK gene pairs being detected between the two species, respectively, with most genes predominantly undergoing purifying selection. Gene expression profiling indicated that NnMAPK cascade genes were extensively involved in plant development and submergence stress response. Co-expression analysis revealed potential interaction between transcription factors (TFs) and NnMAPK cascade genes in various biological processes. NnMKK showed predicted interactions with multiple NnMAP3K or NnMPK proteins, which suggested that functional diversity of MAPK cascade genes could be as a result of their complex protein interaction mechanisms. This first systematic analysis of MAPK cascade families in lotus provides deeper insights into their evolutionary dynamics and functional properties, which potentially could be crucial for lotus genetic improvement.

Tobacco rattle virus-induced VcANS gene silencing in blueberry fruit.

Blueberry (Vaccinium corymbosum L.), a woody perennial bush in the genus Vaccinium, is an economically important and popular fruit crop worldwide. Development the superior cultivars, which including excellent fruit traits, not only means higher yielding and economic efficiency, but also produce fruit that to meet the preferences of different consumers. Excavating fruit quality-related genes, studying their functions, and using transgenic or molecular-assisted breeding are beneficial to the development of excellent blueberry varieties. Genetic transformation is an excellent way to study the function of genes in plants, however, it is a labor-intensive and time-consuming process to genetically transform many woody plants, including blueberry. Virus-induced gene silencing (VIGS) provides an efficient approach to knock-down the expression of target genes for functional analysis. In this study, tobacco rattle virus induced genes silencing (TRV-VIGS) was established in blueberry fruits using the VcANS gene as a reporter. The silenced sector of the skin of blueberry fruits injected with pTRV2 (plasmid Tobacco Rattle Virus, TRV-RNA2)::VcANS remained green or white at 25 days after agroinfiltration. In agroinfiltrated materials, the VcANS transcript levels were much lower in fruits with phenotypic changes (delayed color change) than in those infiltrated with the pTRV2 empty vector. Silencing of VcANS also affected the expression of other genes involved in the anthocyanin synthesis pathway. The experimental results support that VcANS can be used as an effective marker gene for VIGS system. In addition, the TRV-VIGS system has been successfully established in blueberry fruits, which provided an effective verification method for functional identification of unknown genes in blueberry fruits.

The nitrification inhibitor 1,9-decanediol from rice roots promotes root growth in Arabidopsis through involvement of ABA and PIN2-mediated auxin signaling.

1,9-decanediol (1,9-D) is a biological nitrification inhibitor secreted in roots, which effectively inhibits soil nitrifier activity and reduces nitrogen loss from agricultural fields. However, the effects of 1,9-D on plant root growth and the involvement of signaling pathways in the plant response to 1,9-D have not been investigated. Here, we report that 1,9-D, in the 100-400 µM concentration range, promotes primary root length in Arabidopsis seedlings at 3d and 5d, by 10.1%-33.3% and 6.9%-32.6%, and, in a range of 50-200 µM, leads to an increase in the number of lateral roots. 150 µM 1,9-D was found optimum for the positive regulation of root growth. qRT-PCR analysis reveals that 1,9-D can significantly increase AtABA3 gene expression and that a mutation in ABA3 results in insensitivity of root growth to 1,9-D. Moreover, through pharmacological experiments, we show that exogenous addition of ABA (abscisic acid) with 1,9-D enhances primary root length by 23.5%-63.3%, and an exogenous supply of 1,9-D with the ABA inhibitor Flu reduces primary root length by 1.0%-14.3%. Primary root length of the pin2/eir1-1 is shown to be insensitive to both exogenous addition of 1,9-D and ABA, indicating that the auxin carrier PIN2/EIR1 is involved in promotion of root growth by 1,9-D. These results suggest a novel for 1,9-D in regulating plant root growth through ABA and auxin signaling.

Smallholder vegetable farming produces more soil microplastics pollution than large-scale farming.

Microplastics (MPs) accumulation in farmland has attracted global concern. Smallholder farming is the dominant type in China's agriculture. Compared with large-scale farming, smallholder farming is not constrained by restrictive environmental policies and public awareness about pollution. Consequently, the degree to which smallholder farming is associated with MP pollution in soils is largely unknown. Here, we collected soil samples from both smallholder and large-scale vegetable production systems to determine the distribution and characteristics of MPs. MP abundance in vegetable soils was 147.2-2040.4 MP kg-1 (averaged with 500.8 MP kg-1). Soil MP abundance under smallholder cultivation (730.9 MP kg-1) was twice that found under large-scale cultivation (370.7 MP kg-1). MP particle sizes in smallholder and large-scale farming were similar, and were mainly <1 mm. There were also differences in MP characteristics between the two types of vegetable soils: fragments (60%) and fibers (34%) were dominant under smallholder cultivation, while fragments (42%), fibers (42%), and films (11%) were dominant under large-scale cultivation. We observed a significant difference in the abundance of fragments and films under smallholder versus large-scale cultivation; the main components of MPs under smallholder cultivation were PP (34%), PE (28%), and PE-PP (10%), while these were PE (29%), PP (16%), PET (16%), and PE-PP (13%) under large-scale cultivation. By identifying the shape and composition of microplastics, it can be inferred that agricultural films were not the main MP pollution source in vegetable soil. We show that smallholder farming produces more microplastics pollution than large-scale farming in vegetable soil.

Genome-Wide Characterization and Comprehensive Analysis of NAC Transcription Factor Family in Nelumbo nucifera.

NAC (NAM, ATAF, and CUC) is a ubiquitously expressed plant-specific transcription factor (TF) family which is involved in the regulation of various biological processes. However, a systematic characterization of NAC gene family is yet to be reported in lotus. Here, 82 NnNAC genes which included five predicted membrane-bound NAC proteins were identified in the lotus genome. Phylogenetic analysis revealed seven-subfamily clusters (I-VII) of NnNAC proteins, with homologous gene pairs displaying similar conserved motifs and gene structure characteristics. Transactivation assay of NnNAC proteins revealed an extensive transcriptional activation capacity which is mediated by the highly divergent C-terminal activation domain (AD). Expression analysis of NnNAC genes in lotus tissues showed high transcript levels in root, stamen, petal and seed coat. In addition, 30 and 29 differentially expressed NnNAC candidate genes putatively involved in lotus seed development and response to complete submergence stress, respectively, were identified. Overall, our study provides potentially useful candidate gene resources for future molecular breeding of lotus varieties with novel agronomic traits.

Profiling of polyphenols for in-depth understanding of Tartary buckwheat sprouts: Correlation between cultivars and active components, dynamic changes and the effects of ultraviolet B stress.

Tartary buckwheat sprouts have a high nutritional value and are gluten-free, and polyphenols are their main active constituents. However, information regarding the active constituents' difference of Tartary buckwheat sprouts grown from seeds with different morphology, at different developmental stages and environments is limited. Here, we developed a LC-MS-based targeted metabolomics approach to analyze polyphenols (46 flavonoids and 6 anthraquinones) in 40 Tartary buckwheat sprouts varieties. Both flavonoids and anthraquinones contributed to significant differences in sprouts grown from seed with different color or shape. Twenty-seven differential compounds were all at a higher level in 3-day-old sprouts, and the fold change from 3-day-old to 8-day-old sprouts was 1.42-6.64. A total of 25 differential compounds were all significantly upregulated upon UV-B radiation, especially for epicatechin. This study is valuable not only for better breeding cultivars of Tartary buckwheat sprouts, but also assessing their metabolic quality.

Identification and quantification of oligomeric proanthocyanidins, alkaloids, and flavonoids in lotus seeds: A potentially rich source of bioactive compounds.

Untargeted metabolomics was performed to study the profiles of 101 chemicals in lotus seeds using ultrahigh-performance liquid chromatography-photodiode array detection-high-resolution tandem mass spectrometry. Among them, 16 dimeric, 18 trimeric, and 4 tetrameric proanthocyanidins were theoretically identified based on the degree of polymerization, and the number of linkages and the presence of two dihydroflavonols and three glycosylated alkaloids were determined for the first time. The proanthocyanidin, flavonoid, amino acid, and total compound contents were quantified, revealing decreases in their levels during maturation as well as a polymerization process formation of polymers from monomers during seed maturation. Interestingly, glycosylated alkaloids were only detected in seed cotyledons being highest at green-brown stage, whereas proanthocyanidins were present at a concentration of 8,226.19 ± 249.96 µg/g (dry weight) in green-brown stage of seed coats. Our findings may provide insights into the utilization of lotus seeds as a functional food.

Syringic acid from rice roots inhibits soil nitrification and N2O emission under red and paddy soils but not a calcareous soil.

Syringic acid (SA) is a novel biological nitrification inhibitor (BNIs) discovered in rice root exudates with significant inhibition of Nitrosomonas strains. However, the inhibitory effect of SA on nitrification and nitrous oxide (N2O) emissions in different soils and the environmental factors controlling the degree of inhibition have not been studied. Using 14-day microcosm incubation, we investigated the effects of different concentrations of SA on nitrification activity, abundance of ammonia-oxidizing microorganisms, and N2O emissions in three typical agricultural soils. The nitrification inhibitory efficacy of SA was strongest in acidic red soil, followed by weakly acidic paddy soil, with no significant effect in an alkaline calcareous soil. Potential nitrification activity (PNA) were also greatly reduced by SA additions in paddy and red soil. Pearson correlation analysis showed that the inhibitory efficacy of SA might be negatively correlated with soil pH and positively correlated with clay percentage. SA treatments significantly reduced N2O emissions by 69.1-79.3% from paddy soil and by 40.8%-46.4% from red soil, respectively, but no effect was recorded in the calcareous soil. SA addition possessed dual inhibition of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) abundance in paddy and red soil. Structural equation modelling revealed that soil ammonium (NH4 +) and dissolved organic carbon content (DOC) were the key variables explaining AOA and AOB abundance and subsequent N2O emissions. Our results support the potential for the use of the BNI SA in mitigating N2O emissions and enhancing N utilization in red and paddy soils.

Genome-Wide Analysis of Light-Regulated Alternative Splicing in Artemisia annua L.

Artemisinin is currently the most effective ingredient in the treatment of malaria, which is thus of great significance to study the genetic regulation of Artemisia annua. Alternative splicing (AS) is a regulatory process that increases the complexity of transcriptome and proteome. The most common mechanism of alternative splicing (AS) in plant is intron retention (IR). However, little is known about whether the IR isoforms produced by light play roles in regulating biosynthetic pathways. In this work we would explore how the level of AS in A. annua responds to light regulation. We obtained a new dataset of AS by analyzing full-length transcripts using both Illumina- and single molecule real-time (SMRT)-based RNA-seq as well as analyzing AS on various tissues. A total of 5,854 IR isoforms were identified, with IR accounting for the highest proportion (48.48%), affirming that IR is the most common mechanism of AS. We found that the number of up-regulated IR isoforms (1534/1378, blue and red light, respectively) was more than twice that of down-regulated (636/682) after treatment of blue or red light. In the artemisinin biosynthetic pathway, 10 genes produced 16 differentially expressed IR isoforms. This work demonstrated that the differential expression of IR isoforms induced by light has the potential to regulate sesquiterpenoid biosynthesis. This study also provides high accuracy full-length transcripts, which can be a valuable genetic resource for further research of A. annua, including areas of development, breeding, and biosynthesis of active compounds.

Flavonoids and caffeoylquinic acids in Chrysanthemum morifolium Ramat flowers: A potentially rich source of bioactive compounds.

Varieties of chrysanthemums are among the world's most valuable edible ornamental crops. However, the availability and relationship between the bio-chemicals of chrysanthemums and their morphological variations remain unclear. We developed liquid chromatography mass spectrometry to construct a spectral tag library to identify and quantify chemicals of 7 caffeoylquinic acids, 21 flavones and flavonols, 4 carotenoids, and 13 other compounds in 27 cultivars and representative tea of Chrysanthemum morifolium. A correlation analysis found that more acacetin 7-O-galactoside (23) resulted in lighter colored flowers and less acacetin (43) and kaempferol (44) was associated with yellow flowers. Hot-H2O extraction of C. morifolium tea showed that most flavonoids and caffeoylquinic acids dissolved out at 30 min, with 20.977 and 8.958 mg/g GW indicated that C. morifolium, which is used in food and tea, is rich in flavonoids and carotenoids. The results improve our understanding of flavonoid biosynthesis and the mechanisms responsible for flower color.

Specific DNA mini-barcoding for identification of Gekko gecko and its products.

BACKGROUND: The dry body of the Tokay Gecko (Gekko gecko) is the source of a valuable traditional Chinese medicine, it is therefore listed as a Class II protected animal species in China. Due to increasing market demand and a declining supply of the species, a considerable number of adulterants have emerged in the market. Thus, it is necessary to establish an accurate and rapid method of identification for distinguishing G. gecko from its adulterants and for separating it from highly processed products. METHODS: A total of 274 COI sequences were analyzed by using MEGA 5.0 software. Several specific primers were designed to amplify mini-barcode regions and identify G. gecko from its counterfeits and products. RESULTS: 274 COI sequences of G. gecko and 15 adulterants species were analyzed. G. gecko could be distinguished from its adulterants through BLAST analysis, intra- and inter-specific distance analyses, and an NJ tree based on COI sequences. Two pairs of specific primers designed for this study, COISF2/COISR2 and COISF3/COISR3, amplified 200- and 133-bp fragments of the COI region, respectively, both of which were suitable for the identification of G. gecko and its adulterants. Furthermore, COISF3/COISR3 detected G. gecko in 15 batches of products. CONCLUSION: Therefore, the specific DNA mini-barcoding method developed here may be a powerful tool for the identification of G. gecko and counterfeits, and may also be used to distinguish G. gecko from its highly processed by-products.

High Altitude Is Beneficial for Antioxidant Components and Sweetness Accumulation of Rabbiteye Blueberry.

To better understand the effect of growing location on the phytochemical compounds and sensory properties of blueberry (Vaccinium spp.), here we investigated rabbiteye blueberry 'Brightwell' (Vaccinium ashei cv. 'Brightwell') grown in 10 locations of China. Significant differences in terms of total soluble solids, titratable acidity, flavonoids, phenols, as well as proanthocyanidins and anthocyanins, were found in the fruits (berries) of blueberry plants among the different sampled locations. Furthermore, their sensory properties, which evaluated by the electronic tongue method, also significantly differed among the 10 locations. The content of flavonoids, phenols, proanthocyanidins, and anthocyanins all had significant correlations with sensory properties, except that of aftertaste-astringency. A key finding to emerge was that blueberry plants grown at high altitude locations harbored a high content of total soluble solids, flavonoids, phenols, proanthocyanidins, and anthocyanins along with high scores for the sweetness. These results suggested cultivating blueberry at high altitude can produce fruit that not only possess pronounced beneficial health effects but also good taste.

Liquid chromatography-mass spectrometry-based metabolomics analysis of flavonoids and anthraquinones in Fagopyrum tataricum L. Gaertn. (tartary buckwheat) seeds to trace morphological variations.

Polyphenols (flavonoids and anthraquinones) are one of the most important phytochemicals in Fagopyrum tataricum L. Gaertn. (tartary buckwheat). However, the relationship between the polyphenols of tartary buckwheat seeds and their morphological variations is unclear. We developed a liquid chromatography-mass spectrometry-based targeted metabolomics method to study the chemical profiles of 60 flavonoids and 11 anthraquinones in 40 seed cultivars (groats and hulls). Both flavonoids and anthraquinones were related to variations in seed color; the fold change from yellowish-brown to black seeds was 1.24-1.55 in groats and 0.26-0.76 in hulls. Only flavonoids contributed to significant differences in seed shape; the fold change from long to short seeds was 1.29-1.78 in groats and 1.39-1.44 in hulls. Some differential metabolites were identified at higher concentrations in hulls than in groats. This study provides new insights into differences in polyphenols among tartary buckwheat seeds with different color and shape.

Genome-wide analyses reveals a glucosyltransferase involved in rutin and emodin glucoside biosynthesis in tartary buckwheat.

With people's increasing needs for health concern, rutin and emodin in tartary buckwheat have attracted much attention for their antioxidant, anti-diabetic and reducing weight function. However, the biosynthesis of rutin and emodin in tartary buckwheat is still unclear; especially their later glycosylation contributing to make them more stable and soluble is uncovered. Based on tartary buckwheat' genome, the gene structures of 106 UGTs were analyzed; 21 candidate FtUGTs were selected to enzymatic test by comparing their transcript patterns. Among them, FtUGT73BE5 and other 4 FtUGTs were identified to glucosylate flavonol or emodin in vitro; especially rFtUGT73BE5 could catalyze the glucosylation of all tested flavonoids and emodin. Furthermore, the identical in vivo functions of FtUGT73BE5 were demonstrated in tartary buckwheat hairy roots. The transcript profile of FtUGT73BE5 was consistent with the accumulation trend of rutin in plant; this gene may relate to anti-adversity for its transcripts were up-regulated by MeJA, and repressed by ABA.

Characterization and comparison of nitrate fluxes in Tamarix ramosissima and cotton roots under simulated drought conditions.

Tamarix ramosissima Ledeb., a major host plant for the parasitic angiosperm Cistanche tubulosa, and known for its unique drought tolerance, has significant ecological and economic benefits. However, the mechanisms of nitrogen acquisition by the T. ramosissima root system under drought have remained uncharacterized. Here, uptake of nitrate (NO3-) in various regions of the root system was measured in T. ramosissima using Non-invasive Micro-test Technology at the cellular level, and using a 15NO3--enrichment technique at the whole-root level. These results were compared with responses in the model system cotton (Gossypium hirsutum L.). Tamarix ramosissima had lower net NO3- influx and a significantly lower Km (the apparent Michalis-Menten constant; 8.5 µM) for NO3- uptake than cotton under normal conditions. Upon simulated drought conditions, using polyethylene glycol (PEG), NO3- flux in cotton switched from net influx to net efflux, with a substantive peak in the white zone (WZ) of the root. There were no significant NO3- influx signals observed in the WZ of T. ramosissima under control conditions, whereas PEG treatment significantly enhanced NO3- influx in the WZ of T. ramosissima. The effect of PEG application on NO3- fluxes was highly localized, and the increase in net NO3- influx in response to PEG stimulation was also found in C. tubulosa-inoculated T. ramosissima. Consistently, root nitrogen (N) content and root biomass were higher in T. ramosissima than in cotton under PEG treatment. Our study provides insights into NO3- uptake and the influence of C. tubulosa inoculation in T. ramosissima roots during acclimation to PEG-induced drought stress and provides guidelines for silvicultural practice and for breeding of T. ramosissima under coupled conditions of soil drought and N deficiency.