Research On The Movement Mechanism Of The Filaments And Styles Of Amomum Villosum

Amomum villosum Lour.is a perennial evergreen herb classed in the Family of Zingiberaceae and the Genus of Amomum,its dry ripe fruit?Chun Sharen?is one of the four famous South China medicines,and it has the effects of dissipating dampness,promoting appetite,warming the spleen,antidiarrheal,regulating qi,calming fetus and so on.Chun Sharen has been used widely as a traditional Chinese medicine for over 1300years.Due to the special floweret structure of A.villosum——Gynandrium-like,which impedes pollination by insects and other pollinators,the natural seed setting rate of A.villosum is very low,only 1.17%,seriously restricting the development of Chun Sharen industry.In genuine producing area yangchun,farmers improve the production by artificial pollination.Even that,the yield is still less than 5 kg per 666.7 m².Farmer doing artificial pollination is exhausting and damages seedlings,which leads to low yields.Low-income and hard labor inhibit the planting enthusiasm of farmers.If we can change the floweret configuration of the A.villosum,the pollination rate will be enhanced by insects such as honeybees,and thus increase the yield of A.villosum.Increasing pollination rate will be beneficial to expand the production of A.villosum and solve the current market situation in short supply.At the same time,the production cost will be reduced and the income of farmers will be increased.Chen Hong found that the Gynandrium-like structure of A.villosum was caused by the movement of the filament and the style in the development of the floweret of A.villosum.Therefore,in order to lay the foundation for the research on the change of the structure of the floweret and provide the theoretical guidance for improving the natural pollination rate of the A.villosum,the movement mechanism of the filament and the style of A.villosum were studied in this paper based on the research of Chen Hong.The results are as follows:?1?The structure foundation,the pattern and the time of the movement of the filament and the style of A.villosum were studied.According to the division standard of the floweret growth period which established by Chen Hong,the floweret of A.villosum were divided into 0⁷ different growth stages from a 0.5 cm length to a day after flowering.The opening floret is classed in the 6 period,and a day after flowering is classed in the 7 period.The cell size and distribution of the filament and the style at different growth stages were determined by making the paraffin sections.It was found that the movement of the filament was due to the imbalance of cell growth rate on the abaxial and adaxial side of the filament,and this movement took place from the top of the morphology to the bottom of the morphology.In the period of 0 to 3,the cells on the abaxia side of each part of the filaments was larger than that on the adaxial side and exceeded 3.52%to 56.00%;In the fourth and fifth periods,the cells on the adaxial side of the filament sites 4 and 5 were7.15%to 29.84%larger than those on the abaxia side.By the 6th time,the sizes of the cells on the adaxial side of the filament are larger than those of the abaxia side and exceeded2.24%to 21.38%.Therefore,the curvature of the filaments is caused by the imbalance of the rate of expansion of the cells on the abaxial and adaxial side.The bending starts mainly in the 3rd to 4th stages,and the site of movement begins from the part 4 and part 5,and gradually extend toward the bottom of the morphology.The movement of style of A.villosum is caused by the asymmetric distribution of the cells and the imbalance in the growth rate on the abaxial and adaxial side.The structure of the abaxial and adaxial side of the style are asymmetric,with 4 to 6 larger parenchymal cells on the adaxial side than on the abaxial side.In the whole process of floret development,the cells on the adaxial side at the same site were significantly larger than those on the abaxial side.There was no significant difference in the size of the cells at sites1,2 and 3 on the abaxial and adaxial side;and the cells on the adaxial side were 2.10%to45.42%larger than the cells on the abaxial side;the cells on the adaxial side at sites 4 and 5were much larger than the cells on the abaxial side and exceeded 25.36%to 115.99%.From the 3rd to the 4th,the cells at site 5 on the abaxial and adaxial side began to grow rapidly,the cells on the adaxial side increased by 71.67%and the cells on the abaxial side increased by 25.04%;by the 5th to 6th stages,the cells on the adaxial side with a rapid increase of31.92%,the cells on the abaxial side increased by 18.33%,and the rate of increase of the cells on the adaxial side was greater than that of the abaxial side.Therefore,the process of bending and straightening the style is also started from the 3rd to the 4th period,and the part where the movement occurs is mainly at the part 4 and the part 5.The asymmetry of cell morphology on the abaxial and adaxial side is the basis of its movement.The imbalance of cell growth rate is the root cause of its movement.?2?The distribution and relative content of four plant hormones in filaments and styles were studied.The relative contents and distribution characteristics of IAA,GA₃,CTK,ABA in different periods of filaments and styles were determined by immunohistochemistry.From the 3rd to the 4th period,the content of IAA in both the abaxial and adaxial side of the filament has decreased significantly,with the decrease of 52.05%in the adaxial side and 91.34%in the abaxial side.The rate of decrease of IAA content in the abaxial side was much greater than that on the adaxial side,leading the growth rate of the cells on the abaxial side was smaller than that on the adaxial side;the CTK content in the abaxial side decreased by 37.14%,but it was only decreased by 3.74%in the adaxial side,so the cell division ability on the abaxial side rapidly weakened,and the cell division on the adaxial side of the filament was active,and the number of cells increased on the adaxial side;the relative content of GA₃ in the adaxial side increased by 12.02%,but the content of GA₃ in the abaxial side decreased by 7.05%,making the growth rate of the cells on the adaxial side of the filament greater than that of the abaxial side.Therefore,it may be the combined effect of IAA,CTK,and GA₃,which promotes the increase of the volume and the number of the cells on the adaxial side,and finally leads to the beginning of the 3rd to 4th period,the filaments bend from the adaxial side to the abaxial side.During the period from 1 to 3,high concentrations of IAA,GA₃,and ABA on the abaxial side of the style inhibited the increase in cell volume,resulting in smaller cell volume on the abaxial side than on the adaxial side;however,high concentrations of IAA and GA₃ on the abaxial side of the style cooperated with high concentrations of CTK promoted the division of the cells,increased the number of cells on the abaxial side and exceeded the adaxial side,so the style appeared curved from the abaxial side to the adaxial side.From the 3rd to the 4th period,IAA,GA₃,and ABA on the adaxial side decreased to appropriate concentrations,and the inhibitory effect on the growth of the adaxial side cells was relieved,while the IAA and GA₃ on the far axis continued to rise by 21.61%and33.21%,the inhibition of axial cell growth was enhanced;the CTK content at the adaxial side was significantly higher than that at the abaxial side which increased by 12.86%,making the cell division on the adaxial side more active than abaxial side and the number of cells on the adaxial side increasing rapidly;Therefore,it may be the joint action of IAA,CTK,GA₃,ABA,which leads to the increase of the volume and number of the cells on the adaxial side of the style,and the rapid elongation of the style on the adaxial side.So the style moves from the adaxial side to the abaxial side beginning of the 3rd to 4th period,finally change from the curved shape to vertical.?3?The transcriptome data of the filaments and styles was obtained.The total RNA of the filaments and styles from 1 to 7 periods was extracted and then mixed in the equal amounts.After quality control,sequencing was performed using Illumina sequencing technology to obtain the transcriptome data of the filament and style.A total of 94,484 unigenes were obtained,of which a total of 62,174 unigenes were annotated with an annotation rate of 65.73%.There are 1656 unigenes annotations to the phytohormonal signal transduction pathway.There were 329 unigenes annotated to the auxin signal transduction pathway;438 unigenes annotated to the gibberellin signal transduction pathway;245 unigenes annotated to the cytokinin signal transduction pathway;168 unigenes annotated to the abscisic acid signal transduction pathway;191 unigenes annotated to the brassinosteroid signal transduction pathway;176 unigenes annotated to the jasmonic acid signal transduction pathway;74 unigenes annotated to the salicylate signal transduction pathway;139 unigenes annotated to the ethylene signal transduction pathway.Further mining of transcriptome data,42 unigenes were annotated to auxin biosynthesis key enzyme genes;50 unigenes were annotated to gibberellin biosynthesis key enzyme genes;45 unigenes were annotated to key enzymes of cytokinin biosynthesis Genes;a total of 13unigenes were annotated to jasmonic acid biosynthetic key enzyme genes;18 unigenes were annotated to the key genes for brassinolide biosynthesis;29 unigenes were annotated to ABA biosynthetic key enzyme genes;there were 24 unigenes Annotation to the key enzyme genes for salicylic acid biosynthesis;a total of 23 unigenes were annotated to the key enzyme genes for ethylene biosynthesis.?4?The expression patterns in filaments and styles at different growth stages were studied,and the key genes that may function in filament and style movements were screened out.The obtained transcriptomes of filaments and styles of A.villosum were used as reference sequences to perform expression profile sequencing analysis.The total RNA was extracted from the filaments and styles in the period from 1 to 7,and the gene expression profiles of the filament and style at different growth stages were determined by using the BGI-Seq500 after quality control.Concerned about the key enzymes hormone synthesis and key signal transduction factors during 1 to 7 stages in filament and style.Changes in the amount of gene expression and the selection of a large number of genes that may regulate the differential expression of filaments and style bending movements.The third period is the initial period of the occurrence of the movements of the filaments and styles.At the 3rd to 4th stage,AUX1 was up-regulated in filaments,GA₂₀ox,GA₃ox,TCH4,and CYCD3 were down-regulated;AUX1,CKX,B-ARR,GA₂ox,PIF4,CYP85A2,and BKI1 were up-regulated,while TAA1,CPS,GA₃ox,and GID2 were down-regulated.These differentially expressed genes may be key genes regulating the filament and style movements.By analyzing the enrichment of differentially expressed genes in filaments and styles during the same period,it was found that the genes that are differentially expressed in hormonal biosynthesis and signal transduction pathways during the same period include AUX1,GH3,ARF,AUX/IAA,SAUR,and B-ARR,KS,KO,KAO,GA₃ox,GA₂ox,PIF3,GID2,AOC,AOS,JAR1,JAZ,CYP85A2,CYCD3,BRI1,BKI1,TCH4,BAK1,AO,ZEP,NCED,NPR1,PR-1,ACS,ACO,SIMKK,and except KS,the same genes had the same tendency in differential expression in filaments and styles,indicating that these differentially expressed genes play an important regulatory role in the movement of filaments and styles,and this regulation is synchronous.The relative expression levels of 3 gibberellin biosynthetic key enzyme genes and 2gibberellin signal transduction factors in filaments and styles were analyzed by RT-PCR method,it was found that there was a significant positive correlation of the change trend of the relative expressions of GA₃ox,GA₂₀ox,and GA₂ox in the filaments and styles with the change trend of the corresponding gene expression level in the sequencing data of the expression profile.?5?The correlation between the expression levels of key genes of some hormone biosynthesis with the content of corresponding hormones in the filaments and styles were analyzed.Analyzed the correlation between the expression levels of key genes of auxin cytokinins,gibberellins,and abscisic acid biosynthesis enzymes in different growth stages,and the relative amounts of IAA,CTK,GA₃,and ABA in filaments and styles in abaxial and adaxial side,it was found that most of the same key enzyme genes showed extremely significant positive correlations between the expression levels of filaments and styles,indicating that there was a strong correlation between the movement of filaments and styles.The expression of key genes in hormone biosynthesis was positively correlated with the relative content of the hormone ofthe filaments and styles in abaxial and adaxial side,indicating that there is a certain correlation between the expression of key gene of hormone synthesis and the corresponding hormone content during the growth and development of filaments and styles.Summarizing the results of the above studies,the beginning of the filaments and styles movements are all from 3 to 4,and the starting parts of the filament movement are site 4and site 5,and they extend from the morphological upper end to the lower end of morphology;the movement site of style main focus on the upper 4 and 5 morphological sites.The mechanism of bending movement of the filament during the 3rd to 4th period may be as follows:The increase of content of GA₃ inhibited the decrease of IAA and CTK content on the adaxial side of filaments,while the expression of auxin signal transduction factor AUX1 was up-regulated,which promoted the growth of the cells and promoted the elongation of the filaments on the adaxial side.The content of IAA on the abaxial side of filaments rapidly decreased;the GA₃ox and GA₂₀ox genes that are key genes for GA₃biosynthesis were down-regulated,resulting in a decrease in the content of GA₃ on the abaxial side;and on the other hand,the content of CTK on the abaxial side also decreased significantly;The elongation of the filament on the abaxial side.The final result is that the growth rate on the adaxial side of the filaments exceeds the abaxial side,and the bending motion moves from the adaxial side to the abaxial side.The mechanism of movement of the style during the 3rd to 4th period may be as follows:CPS and GA₃ox were down-regulated;GA₂ox,a key enzyme that induces the conversion of active GA to non-biologically active GA,is up-regulated,leading to the content of GA₃ on the adaxial side decreased.Therefore,the inhibitory effect of high concentration of GA₃ on the growth of the adaxial side was relieved;The content of CTK on the paraxial side was increased,at the same time,the expression of B-ARR was up-regulated,which promoted the cleavage of the cells on the adaxial side and the number of cells on the adaxial side was increased.The content of IAA in adaxial side of the style was increased,and the expression of AUX1 was upregulated,which promoted the growth of cells on the adaxial side.The content of GA₃ on the abaxial side increased,and the expression of PIF4,a key factor downstream of the gibberellin signal transduction pathway,was up-regulated,resulting in a high concentration of GA₃ that inhibited the growth of the cells on the abaxial side;at the same time,the content of ABA on the abaxial side was increased,which also inhibits cell division and growth.Therefore,the increase in the number of cells and the rapid increase in cell volume lead to rapid elongation of the adaxial side,while the growth on the adaxial side is inhibited,resulting in the style moving from adaxial side to the abaxial side and eventually becoming straight.