Transcriptomic and metabolomic profiling provide insight into the role of sugars and hormones in leaf senescence of Pinellia ternata.

KEY MESSAGE: The interaction network and pathway map uncover the potential crosstalk between sugar and hormone metabolisms as a possible reason for leaf senescence in P. ternata. Pinellia ternata, an environmentally sensitive medicinal plant, undergoes leaf senescence twice a year, affecting its development and yield. Understanding the potential mechanism that delays leaf senescence could theoretically decrease yield losses. In this study, a typical senescent population model was constructed, and an integrated analysis of transcriptomic and metabolomic profiles of P. ternata was conducted using two early leaf senescence populations and two stay-green populations. The result showed that two key gene modules were associated with leaf senescence which were mainly enriched in sugar and hormone signaling pathways, respectively. A network constructed by unigenes and metabolisms related to the obtained two pathways revealed that several compounds such as D-arabitol and 2MeScZR have a higher significance ranking. In addition, a total of 130 hub genes in this network were categorized into 3 classes based on connectivity. Among them, 34 hub genes were further analyzed through a pathway map, the potential crosstalk between sugar and hormone metabolisms might be an underlying reason of leaf senescence in P. ternata. These findings address the knowledge gap regarding leaf senescence in P. ternata, providing candidate germplasms for molecular breeding and laying theoretical basis for the realization of finely regulated cultivation in future.

Brassinolide as potential rescue agent for Pinellia ternata grown under microplastic condition: Insights into their modulatory role on photosynthesis, redox homeostasis, and AsA-GSH cycling.

Microplastic (MP), as a new pollutant, not only affects the growth and development of plants but also may affect the secondary metabolites of plants. The anti-tumor role of Pinellia ternata is related to secondary metabolites. The role of brassinolide (BR) in regulating plant resistance is currently one of the research hotspots. The paper mainly explores the regulation of BR on growth and physiology of Pinellia ternata under MP stress. The experimental design includes two levels of MP (0, 1%) and two levels of BR (0, 0.1 mg/L). MP led to a marked reduction in plant height (15.0%), Fv/Fm (3.2%), SOD and APX activity (15.0%, 5.1%), whereas induced an evident raise in the rate of O2·- production (29.6%) and GSH content (4.4%), as well as flavonoids (6.8%), alkaloids (75%), and ß-sitosterol (26.5%) contents. Under MP addition, BR supply significantly increased plant height (15.7%), aboveground and underground biomass (16.1%, 10.3%), carotenoid and GSH content (11.8%, 4.2%), Fv/Fm (2.9%), and activities of SOD, GR, and MDHAR (32.2%, 21.08%, 20.9%). These results indicate that MP suppresses the growth of P. ternata, although it promotes secondary metabolism. BR can alleviate the inhibitory effect of MP on growth by improving photosynthesis, redox homeostasis, and the AsA-GSH cycle.

Full-length transcriptome analysis of shade-induced promotion of tuber production in Pinellia ternata.

BACKGROUND: Pinellia ternata is native to China and has been used as a traditional herb due to its antiemetic, antitussive, analgesic, and anxiolytic effects. When exposed to strong light intensity and high temperature during the reproductive growth process, P. ternata withers in a phenomenon known as "sprout tumble", which largely limits tuber production. Shade was previously found to delay sprout tumble formation (STF); however, no information exists regarding this process at the molecular level. Hence, we determined the genes involved in tuber development and STF in P. ternata. RESULTS: Compared to that with natural sun-light (control), shade significantly induced chlorophyll accumulation, increased chlorophyll fluorescence parameters including initial fluorescence, maximal fluorescence, and qP, and dramatically repressed chlorophyll a:b and NPQ. Catalase (CAT) activity was largely induced by shade, and tuber products were largely increased in this environment. Transcriptome profiles of P. ternata grown in natural sun-light and shaded environments were analyzed by a combination of next generation sequencing (NGS) and third generation single-molecule real-time (SMRT) sequencing. Corrections of SMRT long reads based on NGS short reads yielded 136,163 non-redundant transcripts, with an average N50 length of 2578 bp. In total, 6738 deferentially-expressed genes (DEGs) were obtained from the comparisons, specifically D5S vs D5CK, D20S vs D20CK, D20S vs D5S, and D20CK vs D5CK, of which, 6384 DEGs (94.8%) were generated from the D20S vs D20CK comparison. Gene annotation and functional analyses revealed that these genes were related to auxin signal transduction, polysaccharide and sugar metabolism, phenylpropanoid biosynthesis, and photosynthesis. Moreover, the expression of genes enriched in photosynthesis appeared to be significantly altered by shade. The expression patterns of 16 candidate genes were consistent with changes in their transcript abundance as identified by RNA-Seq, and these might contribute to STF and tuber production. CONCLUSION: The full-length transcripts identified in this study have provided a more accurate depiction of P. ternata gene transcription. Further, we identified potential genes involved in STF and tuber growth. Such data could serve as a genetic resource and a foundation for further research on this important traditional herb.

Alteration of crop rotation in continuous Pinellia ternate cropping soils profiled via fungal ITS amplicon sequencing.

Pinellia ternata is a traditional Chinese herb that has been shown to suffer from continuous cropping. Deterioration in continuous P. ternata cropping soils and the effect of alteration of crop rotation on the soil were evaluated by using Illumina high-throughput sequencing technology. Soil physicochemical properties and fungal diversity were significantly lower in continuous P. ternate cropping soil compared with those of uncultivated soil. In contrast, significant increases in soil factors and fungal diversity were detected after the continuous cropping soil was subjected to rotational cropping treatments, indicating a remediation effect of crop rotation on continuous cropping soil. Moreover, with the extension of wheat cropping in the rotations, fungal richness and most physicochemical properties exhibited a significantly increasing trend, suggesting that the remediation effect of crop rotation could be enhanced by extending the cropping of break crops. Furthermore, compared with those of continuous cropping soils, microbes that were potentially beneficial to P. ternate were enriched in the rhizosphere by rotational cropping, whereas micro-organisms that supposedly lead to soil deterioration were suppressed. Redundancy analysis indicated that Podospora and Alternaria were the major microbes that were responsible for increasing soil physicochemical properties; by contrast, the deterioration in soil fertility was mainly attributed to Fusarium micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of deterioration in continuous Pinellia ternate cropping soils and how crop rotations alter the deteriorated soil is still limited, especially regarding the presence of fungi and soil fertility. This research found remediation effects of crop rotation on continuous P. ternate cropping soils and indicates that these effects could be enhanced by extending the cropping of break crops. Community shifts between the continuous and rotational cropping soils and the associated changes in soil fertility will have a considerable impact on the diagnosis of obstacles to continuous P. ternate cropping and enhancement of the remediation effects of crop rotation.

[Effects of Ca2+ on photosynthetic parameters of Pinellia ternata and accumulations of active components in heat stress].

OBJECTIVE: To study the effect of exogenous Ca2+ on photosynthetic parameters of Pinellia ternate and accumulations of active components under high temperature stress. METHOD: The pigment contents of P. ternata leaves, photosynthesis parameters and chlorophyll fluorescence parameters of P. ternata leaves, the contents of guanosine, adenosine and polysaccharide in P. ternata tubers were measured based on different concentrations of exogenous Ca2+ in heat stress when the plant height of P. ternata was around 10 cm. RESULT: The contents of total chlorophyll and ratio of chlorophyll a/b were relatively higher by spaying Ca2+. Compared with the control, spaying 6 mmol x L(-1) Ca2+ significantly enhanced the net photosynthetic rate (Pn), transpiration (Tr) and stomatal limitation (L8), but reduced intercellular CO2 concentration (C) in P. ternata leaves. With the increase of Ca2+ concentration, maximal PS II efficiency (Fv/Fm), actual photosynthetic efficiency (Yield) and photochemical quenching coefficient (qP) initially increased and then decreased, however, minimal fluorescence (Fo) and non-photochemical quenching coefficient (NPQ) went down first and then went up. The contents of guanosine and polysaccharide and dry weight of P. ternata tubers showed a tendency of increase after decrease, and the content of adenosine increased with the increase of Ca2+ concentration. The content of guanosine and polysaccharide in P. ternata tubers and its dry weight reached maximum when spaying 6 mmol x L(-1) Ca2+. CONCLUSION: With the treatment of calcium ion, the inhibition of photosynthesis and the damage of PS II system were relieved in heat stress, which increased the production of P. ternata tubers.

[Effect of NH4(+) -N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism and main chemical composition of Pinellia ternata].

OBJECTIVE: To study the effect of nitrogen forms on nitrogen metabolism and main chemical composition of Pinellia ternate. METHOD: Through the soilless cultivation experiment and based at the same nitrogen level and different NH4(+) -N/NO3(-) -N ratios, nitrate reductase (NR) activity, glutamine synthetase (GS) activity, the content of nitrate nitrogen and ammonium nitrogen in different parts of P. ternate were determined. The contents of total alkaloid, free total organic acids and guanosine in the tuber were determined. The yield of bulbil and tuber was calculated. RESULT: The test results showed that, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of nitrate reductase decreased, the content of nitrate nitrogen in the leaves, petioles and tuber increasing initially, then decreased, and the content of nitrate nitrogen in the root decreased. Meanwhile, with the NH4(+) -N/NO3(-) -N ratio increasing, the activity of glutamine synthetase in the leaves, petioles and root increased, the activity of glutamine synthetase in the tuber increasing initially, then decreased. The contents of ammonium nitrogen in the leaves, tuber and root increased initially, then decreased, and the contents of ammonium nitrogen in the petioles increased with the NH4(+)(-N/NO3(-)-N ratio increasing. The yield of bulbil and tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 75: 25. The content of total alkaloid and guanosine in the tuber were the highest at the NH4(+)-N/NO3(-) -N ratio of 0: 100, and the contents were 0.245% and 0.0197% respectively. With the NH4(+)-N/NO3(-) -N ratio of 50: 50, the content of free total organic acids was the highest, it reached 0.7%, however, the content of free total organic acids was the lowest at the NH4(+) -N/NO3(-) -N ratio of 0: 100. CONCLUSION: Nitrogen fertilization significant influences the nitrogen metabolism, the yield and main chemical composition of P. ternate.

[Effect of light quality on growth and quality of Pinellia ternata].

Measuring the content of soluble reducing sugar, total sugar, soluble protein, guanosine, alkaloids, and succinic acid of Pinellia ternata tuber were measured by anthrone-sulfuric acid colorimetric method, Coomassie brilliant blue method, RP-HPLC, reverse potentiometric titration, acid dye colorimetry, respectively. The result showed that yellow light could promote the growth and development of P. ternata and increase the content of soluble reducing sugar, total sugar, alkaloids, and succinic acid. Under blue light could promote the content of soluble protein and guanosine. Red and yellow light increased the content of chlorophyll a and chlorophyll b, contrastively blue light reduced the content of chlorophyll a and chlorophyll b. White film through the most uniform spectrum was most conducive to the synthesis of chlorophyll a. As single film, blue film, yellow film were more conducive to the synthesis of chlorophyll a, green film and red film had been relatively beneficial to the synthesis of chlorophyll b. Bulbil formed the largest number and the biggest propagation coefficient of P. ternata under red light showed that it could increase the production of P. ternata under red light.

[Endangered resources and origin changes of Pinellia ternate in Huanghuai plain].

The wild Pinellia ternata resource was surveyed by field investigation, visiting surveys combine with literature study and changing factors of the wild P. ternata in the area of Huanghuai Plain were analyzed. The results indicated that Huanghuai Plain wild P. ternata resources were scarce and nearly extinct. The chemical farming methods in Huanghuai Plain have endangered the survival of the wild P. ternata.

Microarray identification of conserved microRNAs in Pinellia pedatisecta.

Large numbers of microRNAs (miRNAs) reportedly play important roles in plant development. However, none has been reported in Pinellia pedatisecta, an important aroid medicinal plant that possesses the only pedate leaf blades and the largest tubers and inflorescences among all Pinellia species. To detect the miRNAs from P. pedatisecta, an in situ synthesized custom miRNA microarray was employed, following the verification for the presence of the miRNAs through reverse transcription polymerase chain reaction (RT-PCR) and the quantitative RT-PCR (qRT-PCR). A total of 99 miRNAs belonging to 22 miRNA families were identified. The RT-PCR was applied to 14 miRNAs detected to validate the microarray results. The qRT-PCR that targeted seven miRNAs showed different expression levels of miRNAs in different tissues. The current research is the first to report on the miRNAs in P. pedatisecta and will enable further investigation of their roles in P. pedatisecta development.

[Genetic transformation of Pinellia ternata with Agrobacterium tumefaciens-mediated sHSP genes].

OBJECTIVE: To establish an efficient genetic transformation system of Pinellia ternata. METHOD: With petioles from test-tube seedlings of P. ternata as explants, Agrobacterium tumefaciens mediation method was adopted to explore the effect of phenolic substances, A. tumefaciens's concentration, infection time, pre-incubation time and co-cultivation time on genetic transformation efficiency of P. ternata. RESULT AND CONCLUSION: The genetic transformation efficiency could be effectively enhanced by infecting in A. tumefaciens culture containing AS 40 mg x L(-1) for 15 min for three days. The petioles were put into the differentiation medium containing 150 mg x L(-1) Kan and 350 mg x L(-1) Carb to screening and cultivation. After around 30 days, microtubers could be observed at both sides of the petioles. Gus staining and PCR verification on the regenerated plants showed that the exogenous gene sHSP had been integrated into genome of P. ternata.

[Study on the virus-free technology of Pinellia temata].

OBJECTIVE: To solve the degradation of production and quality of Pinellia caused by the virus accumulation, rapid propagation technical of virus-free Pinellia was researched. METHODS: Pinellia leaves,petioles as explants, technology of using high temperature (38 degrees C, 40d) and shoot tip culture producing virus-free Pinellia was explored. RESULTS: The results showed that leaves without virus spots was about 88.9% when explants were culture for 40d at high temperature (38 degrees C). 1.0 mg/L 6-BA and 0.1 mg/L NAA could induce seedling from shoot tip,seedling rate is up to 91.4%; MS added 0.5 mg/L 6-BA and 0.1 mg/L NAA was conducive to growth of the plantlets; added 0.5 mg/L KT and 0.5 mg/L NAA was in favor of inducing root and promoting root growth, the survival rate of the transplanting seedling could reach 89.5%. CONCLUSION: A reliable system of virus-free Pinellia propagation is established.

[Effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata].

OBJECTIVE: To study the effect of low temperature on cell synchronization division in suspension culture cells of Pinellia ternata, in order to find a proper condition for cell synchronization. METHOD: The loosened calli of P. ternata were used as materials. The effect of low temperature on cell synchronization division was studied and researched in suspension culture cells by the method of orthogonal experiment, the obtained experimental data was compared and analyzed through the analysis of range and analysis of variance. RESULT: After the low temperature treatment, the cell division index was increased in various degrees, among the total results, when the temperature was set at 8 degrees C for 24 hours and then recovered for 36 hours, the effect was the best, the mitotic index was significantly increased to 11.3%. CONCLUSION: The low temperature could obviously influence the cell synchronization division in suspension culture cells of P. ternata, and the mitotic index was efficiently increased.

[Effect of different fertilizing levels on yields and efficiency components of Pinellia ternata from Sichuan].

OBJECTIVE: The effect of the different fertilizing levels on yields and main active components of Pinellia ternata from Sichuan was studied under the cultivated condition, in order to provide theoretical basis for the standardized cultivation. METHOD: Using one of the wild populations of P. ternate from Sichuan as tested material, the experiment was performed with orthogonal designing methods L25 (5(3)). During growth and development period, agronomic traits such as number of sprouting, inflorescence and bulblets were counted. After harvesting, main chemical compositions, growth and proliferation rates were determined. RESULT: In different fertilizing levels, the P. ternata from Sichuan showed the same growth rhythm, though there were significant difference (P < 0.05) among the average emergence rate,whereas extremely significant difference (P < 0.01) were detected among the average ratio of bolting, the average bulbils, individual growth rate, individual proliferation rate, beta-sitosterol and alkaloid content under different fertilizing levels. Nitrogenous fertilizer that affected the content of alkaloids and beta-sitosterol were extremely significant (P < 0.01), whereas phosphate and potassium fertilizer had no significant effect. The effect of fertilizer factor and inter effects on beta-sitosterol showed no significant effect, but have influence on other indexes. CONCLUSION: The optimum fertilizer composition was 315 kg x hm(-2) of nitrogen, 225 kg x hm(-2) of P2O5 and 270 kg x hm(-2) of K2O.

[Effects of different storage methods on germination and physiological indexes of seed-stem of Pinellia ternata from Sichuan].

OBJECTIVE: To study the optimum storage methods of Pinellia ternata from Sichuan. METHOD: Nine methods were used to overwintering storage of the seed-stem of P. ternata from Sichuan. The germination of seed-stem was observed through the following year after planting, then the germination percentage, vigor of germination and germination index were calculated. At the seedling stage, the physiological indexes like rooting number, root length, root activity, chlorophyll content and catalase activity etc. were determined. RESULT: Under the different storage conditions, all the tested indexes of P. ternata from Sichuan were significantly different between each other (P < 0.05), except the chlorophyll content. CONCLUSION: The seed-stem of P. ternata from Sichuan should be preserved in the wet sand (or in soil) in a greenhouse or 4 degrees C refrigerator. Under these conditions, the germinating vigor and growth activity of seed-stem performed the best.

[Variation of endogenous hormones in formation of microtubers of Pinellia ternata in vitro].

OBJECTIVE: Through analysis of variation and function of five endogenous hormones (IAA, GA3, ABA, ZR and JA) in the formation of microtubers of Pinellia ternata in vitro to explore the physiological and biochemical mechanism of tubers' development. METHOD: The endogenous hormones of microtubers were isolated in different stages of tubers expansion when the leaf stalks of P. ternata were cultured on MS + 6-BA 0.5 mg x L(-1) + NAA 0.1 mg x L(-1) + sucrose 5% medium to induce the formation of microtubers, then them and stems were detected with Enzyme-linked Immunosorbent Assays (ELISA). RESULT: The results showed that the content of IAA, ABA, JA and ZR in the intumescentia period of microtubers were rapidly rising. The curve of GA3 appeared the type of anti-S. CONCLUSION: IAA, ABA, JA, ZR and GA3 played an important role in controlling formation of microtubers in P. ternata.

[Study on influence of arbuscular mycorrhizal fungi Pinellia ternata yield and chemical composition].

OBJECTIVE: To study the influence of arbuscular mycorrhizal fungi to Pinellia ternata leaf index, propagation coefficient, tuber yield and chemical composition. METHOD: The Pinellia ternata leaf length, width, plant height and leafstalk diameter were measured during growing time till P. ternata loded. The leaf index was calculated and the tuber yield were measured after harvest. The contents of guanosine was determined by HPLC method, and the alkaloid contents were determined by spectrophotometric method. RESULT AND CONCLUSION: Inoculated AM fungi could increase P. ternata propagation coefficient and the yield of tuber. Inoculated AM fungi could increase P. ternata leaf index, made leaf tends to be more like bamboo leaf type. Inoculated AM fungi could delayed P. ternata lodging time; G. mosseae + G. intraradices treatment (early inolulation time) proved better influence on increasing fresh weight and dry weight of P. ternata tuber and the propagation coefficient. Inoculated AM fungi could increase guanosine and alkaloid contents of Pinellia ternata tuber, and the contents of guanosine and alkaloid in inoculated treatments were higher than those in cultivated P. ternata from Hezhang; G. mosseae + G. intraradices treatment (later inolulation time) proved good effect on increasing the contents of guanosine and alkaloid.

[Nutrient uptake characteristics of nitrogen, phosphorus and potassium of Pinellia ternate from Sichuan during growth stage].

OBJECTIVE: To study the absorption characteristics of nitrogen (N) , phosphorus (P) , and potassium (K) of Pinellia ternate from Sichuan under the cultivated condition. METHOD: The samples of P. ternate S-29 and S-33 from Sichuan were regularly collected randomly, the contents of nitrogen, phosphorus and potassium of each part (aerial and underground parts) were determined, and their distribution and accumulation characters of nitrogen, phosphorus, potassium were also studied. RESULT: The absorption rate of aerial and underground parts of P. ternate were different in nitrogen, phosphorus, and potassium at different growing stages. Among them, the potassium uptake was the highest, while the phosphorus uptake was the lowest. Totally, the uptakes of nitrogen, phosphorus and potassium of P. ternate from Sichuan during "sprout tumble" stage were comparatively higher than that not at "sprout tumble" stage. A significant correlation was observed between the phosphorus and potassium uptake of the aerial and underground parts, respectively (P < 0.05). And there was also significant positive correlation between the nitrogen and phosphorus uptake at underground parts (P < 0.05). During the growth stage of P. ternate, for producing 100 kg air-dried P. ternate, 5.02-5.64 kg N, 2.44-2.56 kg P205, 11.62-13.49 kg K20 were needed to be absorbed. CONCLUSION: P. ternate from Sichuan should be given enough base fertilizer in field cultivation, and then be supplied with phosphorus and potassium by combining foliar spray with field application during the middle ten days of June and last ten days of August. The ratios of potassium should be properly improved. There was promoting effect in the phosphorus and nitrogen assimilations of the underground parts of P. ternate from Sichuan.

[Effect of original processing method on sterol compounds of rhizoma pinelliae].

OBJECTIVE: Investigate the effect of different processing methods on the quality of Rhizoma Pinelliae. METHODS: The sterol compounds from different Rhizoma Pinelliae samples processed with different drying methods were analysed by GC. RESULTS: The fingerprints of five specific steroid components are very stable in those samples of Rhizoma Pinelliae which were first dried 2 h at 70 degrees C then baked at low temperature or by repeated drying at low-temperature, and the the five peaks from each sample have high similarity. But the samples dried without a proper treatment on higher temperature or with a halfway dryness at low-temperature showed a reduced peak height of steroid to varying degrees and some peaks were hardly detected. CONCLUSION: It is conducive to the preservation and stability of stroids of Pinellia ternata that to being Dried at proper high temperature such as at 70 degrees C for 2 h first then fully dried at low temperature.

[Study on mathematical model of optimum fertilizer application to Pinellia ternate cultivation].

OBJECTIVE: To study the mathematical model of optimum fertilizer application to Pinellia by field experiments. METHOD: Three factors and 5 levels quadratic rotational combination design was applied to study the effects of N, P and K. RESULT AND CONCLUSION: N, P and K combination application can achieve the maximum yield of 29,921.4 kg x hm(-2), the amount of N, P2O5, K2O were 413.79, 224.79, 164.01 kg x hm(-2), respectively. The effects of the 3 factors were in following orders: P > N > K.

[Growth and propagation of wild Pinellia ternata in cultivation].

OBJECTIVE: To study the growth and propagation of Pinella ternata in cultivation. METHOD: Forty six populations of P. ternata originated from Sichuan, Shanxi province and chongqing city were collected and cultivated in experimental field under the same cultivation condition. RESULT AND CONCLUSION: Reproduction tubers at the weight of 0.51-2.00 g (diameter 0.9 - 1.5 cm) had higher increments of tubers and general weight. The P. ternata populations originated from Peng-an county, Shehong county, Zhongjiang county, Lezhi county, Lu county grew faster, on the other hand, the populations originated from Lezhi county, Guangyuan county, Peng-an county, Jianyang county had stronger ability of propagation. The sprouting rate of above 0.30 g (diameter 0.7 cm) tubers was about 90%. There was no significant difference in the growth between tubers and bulbils that have the same weight. The two factors that affected the yield increment were weight range and population, and the former was the main one. Significantly positive correlations were found between weight of reproduction tuber and weight of harvested tubers, number of bulbils. However, significantly negative correlations were found between weight of reproduction tuber and weight increments of tubers, increments proportion of tubers and general weight, and a negative correlations was found between weight of reproduction tuber and general weight increments. Curve-estimated models were conjectured about the weight of reproduction tuber and growth or propagation parameters such as the diameter, the sprouting rate, increments of tubers and general weight, increments proportion of tubers and general weight and the number of bulbils. Some proposals to improve the cultivation of P. ternate were suggested.