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.

Transcriptomic analysis of short-term heat stress response in Pinellia ternata provided novel insights into the improved thermotolerance by spermidine and melatonin.

Heat stress has been a major environmental factor limiting the growth and development of Pinellia ternata which is an important Chinese traditional medicine. It has been reported that spermidine (SPD) and melatonin (MLT) play pivotal roles in modulating heat stress response (HSR). However, the roles of SPD and MLT in HSR of P. ternata, and the potential mechanism is still unknown. Here, exogenous SPD and MLT treatments alleviated heat-induced damages in P. ternata, which was supported by the increased chlorophyll content, OJIP curve, and relative water content, and the decreased malondialdehyde and electrolyte leakage. Then, RNA sequencing between CK (control) and Heat (1 h of heat treatment) was conducted to analyze how genes were in response to short-term heat stress in P. ternata. A total of 14,243 (7870 up- and 6373 down-regulated) unigenes were differentially expressed after 1 h of heat treatment. Bioinformatics analysis revealed heat-responsive genes mainly included heat shock proteins (HSPs), ribosomal proteins, ROS-scavenging enzymes, genes involved in calcium signaling, hormone signaling transduction, photosynthesis, pathogen resistance, and transcription factors such as heat stress transcription factors (HSFs), NACs, WRKYs, and bZIPs. Among them, PtABI5, PtNAC042, PtZIP17, PtSOD1, PtHSF30, PtHSFB2b, PtERF095, PtWRKY75, PtGST1, PtHSP23.2, PtHSP70, and PtLHC1 were significantly regulated by SPD or MLT treatment with same or different trends under heat stress condition, indicating that exogenous application of MLT and SPD might enhance heat tolerance in P. ternata through regulating these genes but may with different regulatory patterns. These findings contributed to the identification of potential genes involved in short-term HSR and the improved thermotolerance by MLT and SPD in P. ternata, which provided important clues for improving thermotolerance of P. ternata.

Remediation of deterioration in microbial structure in continuous Pinellia ternata cropping soil by crop rotation.

Pinellia ternata is a traditional Chinese herb that suffers from continuous cropping (CC), which significantly decreases both yield and quality. The influence of CC on the microbiome in P. ternata rhizosphere and the effects of remediation on microbiota by rotational cropping (CR) were assessed by Illumina high-throughput sequencing technology. CC tends to decrease the α-diversities as a function of cultivation time, whereas CR tends to increase them. Differentially abundant analysis showed that microbial structure was important in maintaining the health status of P. ternata rhizosphere. Results suggested that CC soils were mainly enriched for Pseudomonas, Rhizobium, and Streptophyta operational taxonomic units (OTUs), while the CR soils were mainly enriched for Rhizobium, Pseudomonas, Flavobacterium, Sphingomonas, Rhizobacter, and Arthrobacter OTUs. On the basis of the community dissimilarities, we grouped all sample replicates into three post hoc clusters in which soils were defined as healthy, health-suppressed, and health-depressed soils. The three soil types represented different soil physicochemical properties. The activities of the microbiome features, including ammonia oxidizer, sulfate reducer, nitrite reducer, dehalogenation, xylan degrader, sulfide oxidizer, nitrogen fixation, atrazine metabolism, chitin degradation, degraded aromatic hydrocarbons, and chlorophenol degradation, were also considerably different among the three soils.

[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.

[Protoplasts isolation, purification and plant regeneration of Pinellia cordata].

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

[EMS mutation of suspension cells and selection of high temperature tolerant mutants from Pinellia ternata].

OBJECTIVE: To determine the optimal concentration and processing time of EMS mutation for suspension cells from Pinellia ternata. METHOD: Under four EMS concentration gradients (0.1% , 0.2%, 0.4%, 0.6%) and three processing time gradients (0.5, 1.0, 2.0 h), the suspension cells of P. ternata were mutagenized. RESULT AND CONCLUSION: The results showed that the survival rate was significantly different under the different concentrations of EMS and the different processing time. In the same processing time, the EMS concentrations were increased, but the suspension cells survival rate decreased gradually. The optimum EMS concentration for the mutagenesis was 0.4% and the best processing time was 1 hour.

[Changes of photosynthesis parameters and chlorophyll fluorescence around sprout tumble of Pinellia ternata under high temperature stress].

OBJECTIVE: To study the change of photosynthesis and chlorophyll fluorescence characteristics of Pinellia ternata around sprout tumble. METHOD: Tubers of P. ternata were cultured firstly at (25 +/- 1) degrees C for certain days, and then they were coerced under 37 degrees C stress in the same artificial climate boxes. The chlorophyll contents, photosynthesis and chlorophyll fluorescence during different stages of high temperature stress were measured. RESULT AND CONCLUSION: After under high temperature stress, the chlorophyll contents, the ratio of chlorophyll a/b, photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), maximum fluorescence (Fm), quantum yield of PS II (phi (PS II)), intrinsic photochemical efficiency of PS II (Fv/Fm) and the chlorophyll photochemical quenching (qP) decreased, but the intercellular CO2 concentration (Ci), minimal fluorescence (F0), the chlorophyll nonphotochemical quenching (NPQ) increased.

[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.

[Quality grading of artificial tuber seed of Pinellia ternata].

OBJECTIVE: To establish the seed quality grading standard of artificial tuber of Pinellia ternata. METHOD: Hundred-grain weight, moisture content, germination rate, viability, single-grain diameter, purity and visual form of mature artificial tuber seed from tissue culture of P. ternate were determined. The key indicator and reference indicators for artificial tubers quality grading were defined statistically by SPSS analysis. RESULT: Germination rate and viability were the primary indicators, hundred-grain weight, moisture content and purity were important reference indicators. CONCLUSION: The quality of each grade should reach the following requirements: for first grade, hundred-grain weight > 10 g, moisture content > 70%-80% , germination rate > 80%, viability > 85%, single-grain diameter > 0.7 cm, purity > 80%; for second grade, hundred-grain weight 8-10 g, moisture content > 70%-80%, germination rate 60%-80%, viability 70%-85%, single-grain diameter 0.3-0.7 cm, purity 75%-80%; moisture content > 70%-80%, and the others don't reach second grade was the third grade.

[Isolation and identification of endophytes from Pinellia].

OBJECTIVE: To investigate the varieties of entophytes in different parts of Pinellia ternata. METHOD: The solidified plates were applied for the isolation of the endophytes, and three methods were used for the identification of endophytic fungi. RESULT: Eighty four strains of the entophytes were isolated from the P. ternata collected from 3 habitations. Endophytic fungi were morphologically identified belonging to 15 genera, 4 families. CONCLUSION: It indicated that the entophytes in P. ternata were diversity and rich, and there were some differences at quantity and species in different organs of P. ternata.

[Photosynthetic characteristics and chlorophy II fluorescence parameters of different populations of Pinellia ternata].

OBJECTIVE: To study the photosynthetic characteristics and chlorophyll fluorescence parameters of Pinellia ternate from different sources, in order to provide basis for cultivation and breeding. METHOD: Pigment contents were compared among different sources by the Lichtenthaler method. The light-response and diurnal photosynthesis course of P. ternata from different sources were analyzed by LI-6400 system, and chlorophll fluorescence parameters were analyzed by PAM-2100 system. RESULT: The difference of photosynthetic characteristics among the sources were remarkable. The light compensation point (LCP) of P. ternate from different sources were 11.57-17.88 micromol x m(-2) x s(-1), the light saturation point (LSP) were 1 000-1 300 micromol x m(-2) x (s-1). All the sources have obvious "midday depression". P. ternate cultivated in Xinjiang showed a higher light saturation point (LSP) and better chlorophyll fluorescence parameters. CONCLUSION: P. ternata cultivated in Xinjiang showed better photosynthetic characteristics and chlorophyll fluorescence parameters than the others.

[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.

[Effects of high temperature stress on root vitality and leaf biochemical indexes in different populations of Pinellia ternate].

OBJECTIVE: To study the effects of high temperature stress on the root vitality and leaf biochemical indexes in populations of Pinellia ternate. METHOD: The leaf activity of SOD, contents of MDA and free praline and root vitality were determined after a treatment of high temperature stress (35 degrees C/25 degrees C) was given to different populations of P. ternate. RESULT AND CONCLUSION: By stress time increasing, the leaf SOD and free praline rose firstly and then dropped, the content of MDA increased while the root ability decreased in all populations. And the response of populations of P. ternate to high temperature was significantly different.

[Change of endogenous hormone around sprout tumble of Pinellia ternata under high temperature stress].

OBJECTIVE: To study the change of endogenous hormone (ABA, IAA, JA, GA3, ZR) in the leaves, petioles, tubers of Pinellia ternate around sprout tumble. It also provided some valuable information to prevent sprout tumble and increase production. METHOD: Tubers of P. ternata were cultured firstly at (23 +/- 1) degree C for certain days, and then they were coerced under (30 +/- 1 ) degree C stress in the same artificial climate boxes. The endogenous hormones in leaves, petioles and tubers during different stages of high temperature stress were determined with Enzyme-linked Immunosorbent Assays (ELISA). RESULT: After under high temperature stress, ABA content in leaves, petioles and tubers increased obviously. Similarly, JA content rose all in the leaves, petioles and tubers. But in the same conditions IAA content declined significantly in the leaves and petioles. In the tubers, IAA content also decreased, but not quickly. With the extension of high temperature coercion, the leaves, petioles, tubers, ZR content were gradually falling off. In the leaves of GA3 content rose markedly at the third day, fell down at the sixth day, but remained higher than before treatment. With the extension of the processing time, GA3 content fell off in the petioles and tubers. CONCLUSION: ABA, JA, ZT and GA3 played an important role in controlling sprout tumble of P. ternata.