Effects of Iron Deficiency Stress on Plant Growth and Quality in Flowering Chinese Cabbage and Its Adaptive Response

Iron (Fe) plays an important role in the growth and development of the human body and plants. The effects of different Fe concentrations, 1-aminocyclopropane-1-carboxylic acid (ACC), and cobalt chloride (Co2+) treatments on plant growth, quality and the adaptive response to Fe deficiency stress were investigated in flowering Chinese cabbage. The results revealed that Fe deficiency stress inhibited plant growth. The content of vitamin C (Vc), soluble protein, and soluble sugar in leaves and stalks were significantly reduced under Fe deficiency stress, while the content of cellulose and nitrate was increased. Meanwhile, Fe deficiency stress obviously reduced the net photosynthetic rate and nitrate reductase (NR) activity of leaves. The balance system of active oxygen metabolism was destroyed due to Fe deficiency, resulting in the decrease of catalase (CAT) activity, superoxide dismutase (SOD) activity of roots and leaves, and peroxidase (POD) activity of leaves, while POD activity in roots and malonaldehyde (MDA) content were significantly increased. The treatments of Fe deficiency and ACC significantly reduced pH value of the root medium, promoted release of ethylene, and increased Fe3+ reductase activity, while Co2+ treatment showed the results opposite to those of Fe deficiency and ACC treatments. Thus, Fe deficiency stress could induce nitrogen metabolism, photosynthesis, reactive oxygen metabolism, pH of root medium, and Fe3+ reductase activity that was related to physiological adaptive response and tolerance mechanisms. We also found that ethylene could involve in regulating the adaptive response to Fe deficiency stress and improve the availability of Fe in flowering Chinese cabbage.Main ConclusionFe deficiency stress could induce nitrogen metabolism, photosynthesis, reactive oxygen metabolism, pH of root medium, and Fe3+ reductase activity that was related to physiological adaptive response and tolerance mechanisms.

Identification of DELLA Genes and Key Stage for GA Sensitivity in Bolting and Flowering of Flowering Chinese Cabbage

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) is an important and extensively cultivated vegetable in south China, and its stalk development is mainly regulated by gibberellin (GA). DELLA proteins negatively regulate GA signal transduction and may play an important role in determining bolting and flowering. Nevertheless, no systematic study of the DELLA gene family has been undertaken in flowering Chinese cabbage. In the present study, we found that the two-true-leaf spraying of gibberellin A3 (GA3) did not promote bolting but did promote flowering, whereas the three-true-leaf spraying of GA3 promoted both bolting and flowering. In addition, we identified five DELLA genes in flowering Chinese cabbage. All five proteins contained DELLA, VHYNP, VHIID, and SAW conserved domains. Protein-protein interaction results showed that in the presence of GA3, all five DELLA proteins interacted with BcGID1b (GA-INSENSITIVE DWARF 1b) but not with BcGID1a (GA-INSENSITIVE DWARF 1a) or BcGID1c (GA-INSENSITIVE DWARF 1c). Their expression analysis showed that the DELLA genes exhibited tissue-specific expression, and their reversible expression profiles responded to exogenous GA3 depending on the treatment stage. We also found that the DELLA genes showed distinct expression patterns in the two varieties of flowering Chinese cabbage. BcRGL1 may play a major role in the early bud differentiation process of different varieties, affecting bolting and flowering. Taken together, these results provide a theoretical basis for further dissecting the DELLA regulatory mechanism in the bolting and flowering of flowering Chinese cabbage.

Identification and Analysis of Small Interfering RNAs Associated With Heat Stress in Flowering Chinese Cabbage Using High-Throughput Sequencing

Endogenous small interfering RNAs (siRNAs) are substantial gene regulators in eukaryotes and play key functions in plant development and stress tolerance. Among environmental factors, heat is serious abiotic stress that severely influences the productivity and quality of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). However, how siRNAs are involved in regulating gene expression during heat stress is not fully understood in flowering Chinese cabbage. Combining bioinformatical and next-generation sequencing approaches, we identified heat-responsive siRNAs in four small RNA libraries of flowering Chinese cabbage using leaves collected at 0, 1, 6, and 12 h after a 38°C heat-stress treatment; 536, 816, and 829 siRNAs exhibited substantial differential expression at 1, 6, and 12 h, respectively. Seventy-five upregulated and 69 downregulated differentially expressed siRNAs (DE-siRNAs) were common for the three time points of heat stress. We identified 795 target genes of DE-siRNAs, including serine/threonine-protein kinase SRK2I, CTR1-like, disease resistance protein RML1A-like, and RPP1, which may play a role in regulating heat tolerance. Gene ontology showed that predictive targets of DE-siRNAs may have key roles in the positive regulation of biological processes, organismal processes, responses to temperature stimulus, signaling, and growth and development. These novel results contribute to further understanding how siRNAs modulate the expression of their target genes to control heat tolerance in flowering Chinese cabbage.

Effect of Nitrate Concentration on the Growth, Bolting and Related Gene Expression in Flowering Chinese Cabbage

Nitrogen concentration affects growth and bolting of plants, but its regulation mechanism is still unclear. In this work, three nitrate concentration treatments (5%, 100%, 200%) in nutrient solution were conducted to explore the internal relationship between nitrogen and bolting in flowering Chinese cabbage. The results showed that the bolting and flowering time was earlier under the treatment with low nitrate and, the lower the nitrate concentration, the earlier the bolting and flowering. Low-nitrate treatment reduced the content of nitrate, soluble protein, free amino acid and total nitrogen, and increased the C/N ratio. The C/N ratio was significantly negatively correlated with plant height, stem thickness and biomass, while it was significantly positively correlated with flowering rate. Thus, it was indicated that nitrogen may affect bolting and flowering by regulating the C/N ratio of flowering Chinese cabbage plants. The expression of flowering-related genes (SOC1, LFY) was increased significantly under low nitrate treatment. In addition, the pith cell area at the stem tip was significantly reduced under low nitrate treatment, resulting in a significant decrease in stem thickness. The expression of cyclin- and expansin-related genes (CYCD3-3, CYCB1-1 and EXPA8) was significantly reduced, which indicated that nitrogen may regulate the stem development of flowering Chinese cabbage by regulating the expression of cyclin- and expansin-related genes.

Appropriate NH4+/NO3– Ratio Triggers Plant Growth and Nutrient Uptake of Flowering Chinese Cabbage by Optimizing the pH Value of Nutrient Solution

Compared with sole nitrogen (N), the nutrition mixture of ammonium (NH4+) and nitrate (NO3–) is known to better improve crop yield and quality. However, the mechanism underlying this improvement remains unclear. In the present study, we analyzed the changes in nutrient solution composition, content of different N forms in plant tissues and exudates, and expression of plasma membrane (PM) H+-ATPase genes (HAs) under different NH4+/NO3– ratios (0/100, 10/90, 25/75, 50/50 as control, T1, T2, and T3) in flowering Chinese cabbage. We observed that compared with the control, T1 and T2 increased the economical yield of flowering Chinese cabbage by 1.26- and 1.54-fold, respectively, whereas T3 significantly reduced plant yield. Compared with the control, T1–T3 significantly reduced the NO3– content and increased the NH4+, amino acid, and soluble protein contents of flowering Chinese cabbage to varying extents. T2 significantly increased the N use efficiency (NUE), whereas T3 significantly decreased it to only being 70.25% of that of the control. Owing to the difference in N absorption and utilization among seedlings, the pH value of the nutrient solution differed under different NH4+/NO3– ratios. At harvest, the pH value of T2 was 5.8; in the control and T1, it was approximately 8.0, and in T3 it was only 3.6. We speculated that appropriate NH4+/NO3– ratios may improve N absorption and assimilation and thus promote the growth of flowering Chinese cabbage, owing to the suitable pH value. On the contrary, addition of excessive NH4+ may induce rhizosphere acidification and ammonia toxicity, causing plant growth inhibition. We further analyzed the transcription of PM H+-ATPase genes (HAs). HA1 and HA7 transcription in roots was significantly down-regulated by the addition of the mixture of NH4+ and NO3–, whereas the transcription of HA2, HA9 in roots and HA7, HA8, and HA10 in leaves was sharply up-regulated by the addition of the mixture; the transcription of HA3 was mainly enhanced by the highest ratio of NH4+/NO3–. Our results provide valuable information about the effects of treatments with different NH4+/NO3– ratios on plant growth and N uptake and utilization.

Crosstalk between auxin and gibberellin during stalk elongation in flowering Chinese cabbage

Plant growth and development are tightly regulated by phytohormones. However, little is known about the interaction between auxin and gibberellin acid (GA) during flower stalk elongation and how it is directly related to organ formation. Therefore, the effects of indole acetic acid (IAA) and GA3 treatments and their interaction on flower stalk elongation in flowering Chinese cabbage were investigated. The growth of flowering Chinese cabbage is regulated by IAA and GA3, and the opposite results were observed after treatments with uniconazole (GA synthesis inhibitor) and N-1-naphthylphthalamic acid (NPA) (auxin transport inhibitor). Anatomical analysis of the pith region in stalks revealed that IAA promoted expansion via signal transduction and transport pathways. GA3 regulated the elongation of flower stalks by controlling GA synthesis and partially controlling the IAA signaling pathway. GA3 also had a stronger effect on stalk elongation than IAA. The results of qRT-PCR and histological analysis revealed that GA3 and IAA induced the expansion of cell walls by activating the expression of genes encoding cell wall structural proteins such as Expansin (EXP). These findings provide new insights into the mechanism of stalk formation regulated by the combination of IAA and GA3.

Influence of simulated acid rain on the physiological response of flowering Chinese cabbage and variation of soil nutrients

Flowering Chinese cabbages are widely planted in the south of China and often exposed to acid rain. However, the effect of acid rain on the growth of flowering Chinese cabbage is unclear. In this study, we investigated the influence of simulated acid rain (SAR) on plant height, soil-plant analysis development (SPAD) value (an index for chlorophyll content), proline, malondialdehyde (MDA), antioxidant enzyme activities, nitrogen (N), phosphorus (P), or potassium (K) uptake and variation of soil nutrients. Our results showed that SAR at pH 5.5 did not damage plant development because growth characteristics, photosynthesis, and superoxide dismutase and peroxidase activities did not change obviously at this pH compared to those at pH 7.0. However, 2- to 7-time of SAR exposure at pH 4.5 and pH 3.5 led to the increases of antioxidant enzyme activities, MDA and proline contents, and the decreases of leaf SPAD value and root activity. Nutrient analysis indicated that spraying 4 to 7 times of SAR at pH 3.5 reduced the uptake of N, P and K of flowering Chinese cabbage significantly. In addition, treatment with SAR at pH 3.5 decreased the pH value of the surface soil and the contents of alkaline-hydrolytic N and readily available K but increased that of readily available P in the surface soil by 8.5% to 14.9%. Taken together, our results indicated that SAR at pH 3.5 influenced the antioxidant enzyme system and the contents of soil nutrients, caused metabolic disorders and ultimately restricted the development and growth of flowering Chinese cabbages.

Comparative Analysis of miRNA Expression Profiles between Heat-Tolerant and Heat-Sensitive Genotypes of Flowering Chinese Cabbage Under Heat Stress Using High-Throughput Sequencing

Heat stress disturbs cellular homeostasis, thus usually impairs yield of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). MicroRNAs (miRNAs) play a significant role in plant responses to different stresses by modulating gene expression at the post-transcriptional level. However, the roles that miRNAs and their target genes may play in heat tolerance of flowering Chinese cabbage remain poorly characterized. The current study sequenced six small RNA libraries generated from leaf tissues of flowering Chinese cabbage collected at 0, 6, and 12 h after 38 °C heat treatment, and identified 49 putative novel miRNAs and 43 known miRNAs that differentially expressed between heat-tolerant and heat-sensitive flowering Chinese cabbage. Among them, 14 novel and nine known miRNAs differentially expressed only in the heat-tolerant genotype under heat-stress, therefore, their target genes including disease resistance protein TAO1-like, RPS6, reticuline oxidase-like protein, etc. might play important roles in enhancing heat-tolerance. Gene Ontology (GO) analysis revealed that targets of these differentially expressed miRNAs may play key roles in responses to temperature stimulus, cell part, cellular process, cell, membrane, biological regulation, binding, and catalytic activities. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified their important functions in signal transduction, environmental adaptation, global and overview maps, as well as in stress adaptation and in MAPK signaling pathways such as cell death. These findings provide insight into the functions of the miRNAs in heat stress tolerance of flowering Chinese cabbage.