The primary sources of nitrogen for plants have been suggested to be nitrate (NO3−) and ammonium (NH4+). However, when both nitrate and ammonium are simultaneously available to plants, it is very difficult to differentially quantify NO3−/NH4+ utilization in culture media or soil. Consequently, the contribution of NO3−/NH4+ to total inorganic nitrogen assimilation cannot be determined. We developed a method called the bidirectional stable nitrogen isotope tracer to differentially quantify the nitrate and ammonium utilization by Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets in vitro. The utilization efficiency of nitrate was markedly lower than the utilization efficiency of ammonium for plantlets of both Ov and Bn. In both Ov and Bn, the proportion of NO3−/NH4+ utilization did not show a linear relationship with inorganic nitrogen supply. The Ov plantlets assimilated more nitrate than the Bn plantlets at the lowest inorganic nitrogen concentration. Quantifying the utilization of nitrate and ammonium can reveal the differences in nitrate and ammonium assimilation among plants at different inorganic nitrogen supply levels and provide an alternate way to conveniently optimize the supply of inorganic nitrogen in culture media.

To determine whether the bicarbonate in karst limestone soil could be used as a new carbon resource for biomass production by the catalysis of carbonic anhydrase (CA), a simulative karst drought stress experiment was designed and performed. Three plants used for biofuel energy, Orychophragmus violaceus L. (Ov), Brassica juncea L. (Bj), and Euphorbia lathyris L. (El), were grown under simulated karst drought stress. In response to drought stress, the photosynthesis of the three energy plants was inhibited, but their CA activity increased. The hypothesis was confirmed by plant physiological and stable isotope techniques. The obtained results showed that plant biomass was produced with atmospheric CO₂ as well as bicarbonate under drought stress. Bicarbonate use was proportional to the CA activity of the plants. With high CA activity over a long period, El had the highest proportional bicarbonate use compared to Ov and Bj, reaching 26.95%. Additionally, a new method is proposed for the screening of plants grown for energy in karst habitats.

Three β-type genes coding for carbonic anhydrase and CA activities from Orychophragmus violaceus L. and Brassica juncea L. leaves in response to NaHCO3-induced bicarbonate stress were examined. Three full-length cDNA CDS sequences were designated as OvCA1, OvCA3, and OvCA4 in Orychophragmus violaceus, and as BjCA1, BjCA3, and BjCA4 in Brassica juncea; these genes encoding β-CAs were identified and characterized. In particular, OvCA1 and BjCA1 encode two putative chloroplast isoforms. OvCA3 and BjCA3 encode two putative cytosolic isoforms. OvCA4 and BjCA4 encode two putative plasma membrane isoforms. Quantitative real-time RT-PCR analysis revealed that OvCA1 and OvCA4 expressions in Orychophragmus violaceus, BjCA1, and BjCA4 expressions in Brassica juncea changed synchronously with CA activities as bicarbonate stress was intensified. Bicarbonate stress synchronously stimulated OvCA1 and OvCA4 expressions along with CA activities in Orychophragmus violaceus at slight stress level; but it decreased CA activity, BjCA1 and BjCA4 expressions, and stimulated BjCA3 expression in Brassica juncea. Orychoophragmus violaceus could better adapt to slight bicarbonate stress than Brassica juncea due to the former exhibiting higher OvCA3 expression levels and CA activities than the latter. The responses of CA1 and CA4 in Orychophragmus violaceus and CA3 in Brassica juncea to bicarbonate stress partly regulate HCO3 - to water and CO2 supplied to plants. Diverse CA gene expressions can partially account for different adaptation strategies of the two plant species subjected to different bicarbonate stress levels.

Ammonium (NH₄⁺) toxicity impairs plant growth, but nitrate (NO₃⁻) can mitigate this effect through unresolved mechanisms. Using leaf δ¹³C values (photosynthetic capacity) and a bidirectional ¹⁵N tracer (NH₄⁺ assimilation efficiency and source utilization), this study investigated these mechanisms in 35-day-old Orychophragmus violaceus plantlets grown in modified Murashige and Skoog media under varying NH₄⁺:NO₃⁻ ratios. ¹⁵N isotope fractionation during NH₄⁺ (same fixed 20 mM NH₄Cl) assimilation decreased with increasing NO₃⁻ supply (10, 20, and 40 mM NaNO₃). Under 20 mM NH₄⁺ (δ¹⁵N = −2.64‰) at two ¹⁵NO₃⁻-labels (δ¹⁵N-NO₃⁻ = 8.08‰, low ¹⁵N, L) and (δ¹⁵N-NO₃⁻ = 22.67‰, high ¹⁵N, H), increasing NO₃⁻ concentrations enhanced NO₃⁻ assimilation, alleviating acidic stress from NH₄⁺ and improving photosynthesis. Higher NO₃⁻ levels also increased NH₄⁺ utilization efficiency, reducing futile NH₄⁺ cycling and decreasing associated ¹⁵N fractionation during assimilation. Our results demonstrate that NO₃⁻ alleviates NH₄⁺ toxicity primarily by enhancing photosynthetic performance and optimizing NH₄⁺ utilization efficiency.

Alfalfa ( Medicago sativa L.) is often constrained by factors such as drought and salinity-alkalinity in cultivation. This study aims to investigate the differential response characteristics and physiological-ecological mechanisms of alfalfa during the germination stage under drought, bicarbonate, and drought-salt combined stress. Drought stress was simulated using PEG-6000 (0–20%), and salinity-alkalinity stress was simulated using NaHCO ₃ (0–30 mM) to conduct germination tests on 12 alfalfa varieties. Based on the semi-inhibitory concentrations of germination and phenotypic indicators of the 12 alfalfa varieties under either drought or bicarbonate stress, drought-salt combined stress was applied. A membership function method was used to comprehensively evaluate the differential responses of the 12 alfalfa varieties to drought and salinity during germination, as well as the physiological-ecological response mechanisms of drought-tolerant, salt-tolerant, and drought-salt sensitive varieties. (1) There were significant differences among varieties in response to single drought or bicarbonate, identifying drought-tolerant varieties WL440HQ and WL363HQ, salt-tolerant variety WL525HQ, drought-sensitive 30°N, and salt-sensitive WL343HQ. Comprehensive evaluation indicated that the strongest and weakest drought and salt-resistant varieties were WL363HQ and WL319HQ, respectively; (2) Drought-salt combined stress exhibited antagonism at low concentrations and synergism at high concentrations; (3) The drought-salt tolerant variety WL363HQ adapted well to the combined stress through mechanisms involving SOD activity and soluble sugars, while the sensitive variety WL319HQ performed poorly. This study provides a scientific basis for elucidating the germination response mechanisms of plants under drought-salt combined stress and offers scientific support for planting in arid and saline-alkaline regions based on the selected drought- and salt-tolerant varieties.

In order to obtain the genetic basis of transcriptome data of Orychophragmus violaceus seedlings, the transcriptome of Orychophragmus violaceus was paired-end sequenced by Illumina Novaseq 6000 platform, a total of 59174171 clean reads (17.75 Gb clean bases) were obtained, and 110919 unigenes were obtained after assembly by de novo, with the longest and shortest length of 15030, 301 bp and an average length of 784 bp. The N50 was 947 bp and the N90 was 396 bp. These unigenes were compared among seven public databases including Non-redundant protein sequences (NR), Nucleotide (NT), Swiss-prot protein database (Swiss-Prot), Protein family (Pfam), Eu-karyotic ortholog groups (KOG), Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG), as a result of 75369 (67.94%), 69004 (62.21%), 62258 (56.12%), 56068 (50.54%), 27796 (25.05%), 56066 (50.54%), 32897 (29.65%) unigenes were annotated respectively. These annotation results showed that Orychophragmus violaceus had most homologous sequences with 13610 unigenes with Quercus suber. The GO annotations showed that 56066 unigenes were annotated with 219038, which were divided into 3 categories and 43 functional groups. The KOG annotations showed that 27796 unigenes were annotated and grouped into 25 functional categories. The KEGG annotations showed that 32897 unigenes were involved in 34 types of metabolic pathways and 305 metabolic pathway branches. A total of 18118 SSR sites and 112584 CDS sequences were detected according to analyzing the coding sequences and microsatellite. Base on the high-throughput transcriptome sequencing of Orychophragmus violaceus, with a large number of functional genes are excavated, which provide certain basic data support for the subsequent development of bioinformatics analysis such as molecular markers and functional metabolic pathways.