Exploring the dynamic adaptive responses of Epimedium pubescens to phosphorus deficiency by Integrated transcriptome and miRNA analysis.

Phosphorus, a crucial macronutrient essential for plant growth and development. Due to widespread phosphorus deficiency in soils, phosphorus deficiency stress has become one of the major abiotic stresses that plants encounter. Despite the evolution of adaptive mechanisms in plants to address phosphorus deficiency, the specific strategies employed by species such as Epimedium pubescens remain elusive. Therefore, this study observed the changes in the growth, physiological reponses, and active components accumulation in E. pubescensunder phosphorus deficiency treatment, and integrated transcriptome and miRNA analysis, so as to offer comprehensive insights into the adaptive mechanisms employed by E. pubescens in response to phosphorus deficiency across various stages of phosphorus treatment. Remarkably, our findings indicate that phosphorus deficiency induces root growth stimulation in E. pubescens, while concurrently inhibiting the growth of leaves, which are of medicinal value. Surprisingly, this stressful condition results in an augmented accumulation of active components in the leaves. During the early stages (30 days), leaves respond by upregulating genes associated with carbon metabolism, flavonoid biosynthesis, and hormone signaling. This adaptive response facilitates energy production, ROS scavenging, and morphological adjustments to cope with short-term phosphorus deficiency and sustain its growth. As time progresses (90 days), the expression of genes related to phosphorus cycling and recycling in leaves is upregulated, and transcriptional and post-transcriptional regulation (miRNA regulation and protein modification) is enhanced. Simultaneously, plant growth is further suppressed, and it gradually begins to discard and decompose leaves to resist the challenges of long-term phosphorus deficiency stress and sustain survival. In conclusion, our study deeply and comprehensively reveals adaptive strategies utilized by E. pubescens in response to phosphorus deficiency, demonstrating its resilience and thriving potential under stressful conditions. Furthermore, it provides valuable information on potential target genes for the cultivation of E. pubescens genotypes tolerant to low phosphorus.

Identification of genes involved in metabolism and signalling of abscisic acid and gibberellins during Epimedium pseudowushanense B.L.Guo seed morphophysiological dormancy.

KEY MESSAGE: Key genes involved in metabolism and signalling of abscisic acid and gibberellins during Epimedium pseudowushanense B.L.Guo seed morphophysiological dormancy release were identified using phytochemistry, transcriptomics, and bioinformatic methods. The molecular mechanism of seed morphophysiological dormancy of Epimedium pseudowushanense B.L.Guo. remains largely unknown. The endogenous abscisic acid (ABA) and gibberellin (GA) content of E. pseudowushanense seeds at three developmental stages were quantitatively determined. The results showed the levels of ABA in E. pseudowushanense seeds decreased during seed embryo growth and development, while levels of GA3 increased during seed embryo growth, and levels of GA4 increased during seed dormancy release and seed sprouting. A high-throughput sequencing method was used to determine the E. pseudowushanense seed transcriptome. The transcriptome data were assembled as 178,613 unigenes and the numbers of differentially expressed unigenes between the seed development stages were compared. Computer analysis of reference pathways revealed that 12 candidate genes were likely to be involved in metabolism and signalling of ABA and GAs. The expression patterns of these genes were revealed by real-time quantitative PCR. Phylogenetic relationships among the deduced E. pseudowushanense proteins and their homologous proteins in other plant species were analysed. The results indicated that EpNCED1, EpNCED2, EpCYP707A1, and EpCYP707A2 are likely to be involved in ABA biosynthesis and catabolism. EpSnRK2 is likely implicated in ABA signalling during seed dormancy. EpGA3ox is likely to be involved in GA biosynthesis. EpDELLA1 and EpDELLA2 are likely implicated in GA signalling. This study is the first to provide the E. pseudowushanense seed transcriptome and the key genes involved in metabolism and signalling of ABA and GAs, and it is valuable for studies on the mechanism of seed morphophysiological dormancy.

Pollen competition between morphs in a pollen-color dimorphic herb and the loss of phenotypic polymorphism within populations.

Flower color polymorphism is relatively uncommon in natural flowering plants, suggesting that maintenance of different color morphs within populations is difficult. To address the selective mechanisms shaping pollen-color dimorphism, pollinator preferences and reproductive performance were studied over three years in Epimedium pubescens in which some populations had plants with either green or yellow pollen (and anthers). Visitation rate and pollen removal and receipt by the bee pollinator (Andrena emeishanica) did not differ between the two color morphs. Compared to the green morph, siring success of the yellow morph's pollen was lower, but that of mixtures of pollen from green and yellow morphs was lowest. This difference, corresponding to in vivo and ex vivo experiments on pollen performance, indicated that pollen germination, rather than tube growth, of the green morph was higher than that of the yellow morph and was seriously constrained in both morphs if a pollen competitor was present. A rare green morph may invade a yellow-morph population, but the coexistence of pollen color variants is complicated by the reduced siring success of mixed pollinations. Potential pollen competition between morphs may have discouraged the maintenance of multiple phenotypes within populations, a cryptic mechanism of competitive exclusion.

[Research on photosynthetic and eco-physiological characteristics of Epimedium pseudowushannense in different growing age].

Epimedium is a widely used medicinal plants. Due to excessive use of wild resources and resource constraints, artificial cultivation is the only way to achieve sustainable use of resources. E. pseudowushannense is intended to achieve the first cultivated species, but plant production technology is not yet fully mature.The diurnal variation of photosynthetic and eco-physiological characteristics of E. pseudowushannense in different growing age was studied by the CI-340 Portable Photosynthesis System under natural conditions. The net photosynthetic rate (Pn), stomatal conductance (C), intercellular CO2 concentration (Ci), photosynthetically active radiation (PAR), relative humidity (RH), air temperature (Ta), apparent mesophyll conductance (ALMC), transpiration rate (E), leaf temperature (Tl), water use efficiency (WUE) and other photosynthetic physiological factors and environmental factors were measured. The obtained data was analyzed by correlation analysis, path analysis and stepwise multiple-regression analysis to explore the relationship between net photosynthetic rate and physiological and ecological factors. The results show that： ①The daily variation of the Pn, E, ALMC, C of E. pseudowushannense in different growing age took on"two peak type", the Pn was with an obvious midday depression phenomenon at noon.②The Pn, ALMC and C of annuals' were significantly higher than biennial or three-year; but the Ci was basically a three-year > biennial> annual.③The correlation analysis, path analysis and stepwise regression analysis showed that ALMC, Ci, Ta might played important role in the Pn and ranked as ALMC>Ci>Ta.

[Effects of fluridone, gibberellin acid and germination temperature on dormancy-breaking for Epimedium wushanense].

We introduced Epimedium wushanense seed which has been stratified for 90 days at 10/20 ℃ as experimental materials, with which we studied the effects of fluridone, gibberellin acid and temperature on E. wushanense germination. The results were suggested as shown below. ①Temperature, fluridone and gibberellin acid can both solely or jointly affect germination energy, germination rate significantly. Among those factors, fluridone affect germination rate and germination energy the most, followed by gibberellin acid and temperature. The highest germination rate under 4 ℃ and 10/20 ℃ stratification are 79.3%, 72.0% respectively, which resulted from treatment of F10GA300 and F20GA200 respectively. The highest germination energy under 4 ℃ and 10/20 ℃ stratification are 52.7%, 52.0%, respectively, which both resulted from F20GA200. ②Compared with 4 ℃ germination, seed could not germinate at 10/20 ℃ germination. Nontheless, application of fluridone can lead E. wushanense seeds to germinating.③The effects of gibberellin acid and interaction between gibberellin acid and fluridone significantly affect seed rotten rate during germination. In addition, soaking is another remarkable factor which increased seed rotten rate. As a result, it is feasible to promote E. wushanense dormancy releasing with gibberellin acid and fluridone associating with a proper germination temperature. Further, it is necessary taking actions to avoid seed rotten rate for saving E. wushanense nurseries'cost.

Study on the differences of major pharmaceutical ingredients in different parts and processed medicinal material of Epimedium Brevicornu Maxim in Taihang mountain / Estudio sobre las diferencias de ingredientes farmacéuticos mayor en diferentes partes y procesado medicinal material de epimedium brevicornu maxim en taihang montaña

The aim of this study is to evaluate the medicinal values of different parts of Epimedium brevicornu Maxim. and the effect of processing on major pharmaceutical ingredients in it. The contents of icariin and epimedin C in different parts and processed medicinal material of E. brevicornu in Taihang Mountain were determined with ultrasonic extraction and RP-HPLC. The results indicated that the contents of icariin and epimedin C, respectively 3.4524% and 0.5485%, in the leaf are higher than that in other parts. The contents of icariin and epimedin C, respectively 0.1942 % and 0.1342%, in the stem (include petiole) are the lowest. The contents of these ingredients in the root (include rhizome) are close to that in the leaf. The icariin and epimedin C in all parts of E. brevicornu reduced after processing. The content of icariin in the processed leaf is about 59.5% of that in unprocessed leaves. The effect of prossing on the content of icariin in the stem is unobvious. The content of epimedin C in the processed leaf is about 33.7% of that in unprocessed leaf. The content of epimedin C in the processed stem (include petiole) is about 36.9% of that in unprocessed stem. It is worth to exploit the stem and petiole of E. brevicornu because there are certain contents of pharmaceutical ingredients in them. The firepower should be paid attention to and the temperature should not be very high to avoid the damage on pharmaceutical ingredients in E. brevicornu when process it (AU)

Este estudio pretende evaluar los valores medicinales de diferentes partes de la Epimedium brevicornu Maxim y el efecto de su procesamiento sobre sus principales componentes farmacéuticos. El contenido de icariina y epimedin C en diferentes partes y en material medicinal procesado de Epimedium brevicornu en la montaña de Taihang fue determinado mediante extracción ultrasónica y RP-HPLC. Los resultados indicaron que el contenido de icariina y epimedin C, respectivamente 3,4524% y 0,5485%, en la hoja son mayores que en otras partes. El contenido de icariina y epimedin C, respectivamente 0,1942% y 0,1342%, en el tallo (peciolo incluido) es más bajo. El contenido de estos componentes en la raíz (rizoma incluido) es similar al de la hoja. El contenido de icariina y epimedin C en todas las partes de E. brevicornu se vio reducido después del procesado. El contenido de icariina en la hoja procesada es aproximadamente el 59,5% del de la hoja sin procesar. El efecto del procesado sobre el contenido de icariina en el tallo no es evidente. El contenido de epimedin C en el tallo procesado es de aproximadamente el 33,7% del de la hoja sin procesar. El contenido de epimedin C en el tallo procesado (peciolo incluido) es de aproximadamente el 36,9% de aquel del tallo sin procesar. Vale la pena aprovechar el tallo y peciolo de la E. brevicornu porque hay cierto contenido de componentes farmacéuticos en ellos. Hay que controlar la potencia de fuego y la temperatura no debe ser muy alta para evitar dañar los componentes farmacéuticos de la E. brevicornu (AU)

Pollen packaging and dispensing: adaption of patterns of anther dehiscence and flowering traits to pollination in three Epimedium species.

Pollen presentation theory (PPT) predicts that plant species typically pollinated by frequent and wasteful pollinators ought to be much more parsimonious and only gradually release pollen compared to plant species pollinated by infrequent pollinators that are efficient at delivering the pollen they remove. To test PPT, we compare the pollen presentation schedules and pollination systems in three related Epimedium species, having different pollinators. Results showed that differences in anther dehiscence and flowering traits resulted in different pollen packaging schedules. For E. sutchuenense and E. franchetii, a special 'roll-up' movement of the anther wall during anther dehiscence increased pollen removal compared to the dehiscence pattern in E. mikinorii, which lacked the 'roll-up' movement. Investigations revealed that honeybees had a higher pollen removal rate and lower stigmatic pollen load compared to bumblebees. In accordance with PPT, E. sutchuenense presents pollen sequentially and slowly for the frequent and wasteful honeybees. In comparison to E. sutchuenense, E. franchetii had a faster presentation rate and was adapted to the efficient and infrequent bumblebees. However, E. mikinorii was pollinated by both bumblebees and honeybees at high frequency and had the fastest pollen presentation. This pattern could reduce pollen wastage by honeybees and might be an adaptation to its short flower longevity (less than 1 day), to increase the chances of pollen deposition on stigmas. The study indicates that pollen presentation schedules can be a consequence of interactions among anther dehiscence, flowering traits and pollination environments for a given species.

The differential contributions of herkogamy and dichogamy as mechanisms of avoiding self-interference in four self-incompatible Epimedium species.

Self-interference is one of the most important selective forces in shaping floral evolution. Herkogamy and dichogamy both can achieve reductions in the extent of self-interference, but they may have different roles in minimizing self-interference in a single species. We used four self-incompatible Epimedium species to explore the roles of herkogamy and dichogamy in avoiding self-interference and to test the hypothesis that herkogamy and dichogamy may be separated and become selected preferentially in the taxa. Two species (E. franchetii and E. mikinorii) expressed strong herkogamy and weak protogyny (adichogamy), whereas another two species (E. sutchuenense and E. leptorrhizum) expressed slight herkogamy and partial protandry. Field investigations indicated that there was no physical self-interference between male function and female function regarding pollen removal and pollen deposition in all species. Self-pollination (autonomous or facilitated) was greater in species with slight herkogamy than in those with strong herkogamy. Artificial pollination treatments revealed that self-pollination could reduce outcrossed female fertility in all species, and we found evidence that self-interference reduced seed set in E. sutchuenense and E. leptorrhizum in the field, but not in E. franchetii and E. mikinorii. These results indicate that well-developed herkogamy is more effective compared with dichogamy in avoiding self-interference in the four species. In genus Epimedium, herkogamy instead of dichogamy should be selected preferentially and evolved as an effective mechanism for avoiding self-interference and might not need to evolve linked with dichogamy.

[Research on growth state and physiological changes of Epimedium wushanense under different shade conditions].

OBJECTIVE: To study the growth state and physiological changes of Epimedium wushanense under different light conditions (100%, 80%, 60%, 40%, 20%), and investigate its adaptation mechanism to shade environment so that it can provide theoretical basis for cultivation and production. METHOD: The growth index, the content of photosynthetic pigments, proline content, MDA content, soluble sugar content, soluble protein content and antioxidant enzymy activity of E. wushanense under different shade conditions were measured. RESULT: Except for length/width, the other form index of E. wushanense changed significantly. The E. wushanense grew well between light intensity 80%-40%, besides, the biomass above the earth was relatively high. The chlorophyll a, chlorophyll b, total chlorophyll content and carotenoids cotent were enhanced after shaded, the values of chlorophyll a\chlorophyll b increased between light intensity 100% -60% and reduced between light intensity 60% -20%. The proline and MDA content decreased after shading, the soluble sugar and soluble protein content increased respectively between light intensity 100% -60%, 100% -40% and reduced respectively between light intensity 60% -20%, 40% -20%. The SOD activity, CAT activity decreased after shading, and the POD activity, APX activity reduced between light intensity 60% -40%, raised under 20% treatment. CONCLUSION: 80% 40% irradiance treatment was favorable to the growth of E. wushanense.

[Dormancy characteristics and breaking method of seeds from Epimedium wushanense].

OBJECTIVE: To research the mechanism of dormancy and find out the breaking method for the seeds of Epimedium wushanense. METHOD: The water permeability of seed coat was tested by weighing seeds. The germination inhibitor of the seeds were determined with biotic measurement. The development of embryos, germination rate and germination potential were determined after stratification. RESULT: The water permeability of seed coat was 41.86% after 5 h. The extracts of seeds had strong inhibition effects to the length growth of cabbage seedlings. The growth and development of embryos under the cold stratification (5 degrees C) were better than that under the other conditions. The embryo rate extended from 15.39% to 86.21% after 90 d. Germination rate and germination potential after stratification under 5 degrees C were significantly higher than that under other temperatures. CONCLUSION: The results showed that there was no obstacle of water permeability on the test of E. wushanense, after-ripening of embryogenesis and the germination inhibitor of the seed were the main reason for the seed dormancy. The cold stratification would be an effective way for breaking of the dormancy, which could significantly promote the seed embryogenesis and increase germination rate comparing to other methods.