Mitochondrial pathway of programmed cell death in Paeonia lactiflora pollen cryopreservation.

Programmed cell death (PCD) is an important factor to reduces the viability of plant germplasm after cryopreservation. However, the pathways by which PCD occurs is not fully understood. To investigate whether there is a mitochondrial pathway for pollen PCD after cryopreservation, the pollen of Paeonia lactiflora two cultivars with different PCD levels after cryopreservation was used as test material and the changes of mitochondrial calcium ions (Ca2+), structure, function and their relationship with PCD were compared. The results showed that compared with fresh pollen, the PCD of 'Feng Huang Nie Pan' was significantly reduced after cryopreservation. Their mitochondrial Ca2+ content decreased by 74.27%, mitochondrial permeability transition pore (MPTP) opening reduced by 25.41%, mitochondrial membrane potential slightly decreased by 5.02%, cardiolipin oxidation decreased by 65.31%, and oxygen consumption remained stable, with a slightly ATP production increase. On the contrary, compared with fresh pollen, 'Zi Feng Chao Yang' showed severe PCD after cryopreservation. The decline in mitochondrial Ca2+-ATPase activity led to an accumulation of excessive Ca2+ within mitochondria, triggering widespread opening of MPTP, significantly affecting mitochondrial respiration and energy synthesis. These results suggest the mitochondrial pathway of PCD exists in pollen cryopreservation.

Exploring the relationship between pollen viability and inclusion in Paeonia lactiflora after cryopreservation.

Pollen, as the male gametophyte, carries half of plant genetic information and is an important source of germplasm. The cryopreservation of pollen can not only preserve germplasm, but also solve the problem of time and space barrier in crossbreeding. So it is of great significance to explore the mechanism of pollen viability maintenance after cryopreservation. In this paper, 10 cultivars of Paeonia lactiflora with different fresh pollen viability that did not change after cryopreservation were taken as objects and the effects of pollen inclusions such as soluble sugar, starch, soluble protein, free amino acids, and proline were explored. The results showed that: (1) The contents of pollen inclusions in the fresh pollen of 10 cultivars were different. After cryopreservation, the contents of starch and free amino acids significantly decreased in 10 cultivars, and the soluble sugar, soluble protein, and proline varied with cultivars. (2) Correlation analysis showed that fresh pollen viability was significantly positively correlated with the soluble sugar (R-values of 0.630) and starch content (R-values of 0.694) in fresh pollen. But after cryopreservation pollen viability was only significantly positively correlated with the starch content (R-values of 0.725). These results suggest that the effects of pollen inclusions on pollen vitality are different before and after cryopreservation. The fresh pollen with higher soluble sugar and starch is more vital. But after cryopreservation, the pollen with high starch content has higher viability. The maintenance of stable pollen viability after cryopreservation appears to be related to starch content or starch metabolism, which requires further to study for a final determination.

Effect of the pectin contents and nanostructure on the stem straightness of two Paeonia lactiflora cultivars.

Herbaceous peony (Paeonia lactiflora Pall.) is an ancient ornamental crop and, in recent decades, an emerging popular cut flower. Straight stems are a vital criterion for cut herbaceous peony selection, while many cultivars bend as the plant develops. Pectin helps maintain the mechanical strength of the cell wall. However, little is known about its role in the stem bending of herbaceous peony. Two herbaceous peony cultivars with contrasting stem morphologies ('Dong Fang Shao Nv', upright; 'Lan Tian Piao Xiang', bending gradually) at five developmental stages were used as materials to investigate the effects of pectin content and nanostructure on straightness using the carbazole colorimetric method and atomic force microscopy observations. The contents of water-soluble pectin (WSP), CDTA-soluble pectin (CSP), and sodium carbonate-soluble pectin (SSP) differed significantly between the two cultivars, and the contents and angle of the flower and branch showed correlations. For the pectin nanostructure, WSP showed agglomerates and long chains, with a higher proportion of broad agglomerates at the later stages of the bending cultivar than the upright cultivar. CSP showed branched chains, and the proportion of broad chains was higher in the upright cultivar at later stages, while CSP shape changed from agglomerates to chains in the bending cultivar. SSP mainly consisted of short linear main chains, and side chains in the upright stem were stacked, and the bent cultivar had more broad and short chains. It can be concluded that the contents, nanometric shape, and size of the three kinds of pectin are highly likely to affect herbaceous peony stem straightness. This study provides a theoretical basis for the role of pectin in the production and breeding of herbaceous peony cut flowers.

Xylem development and phloem conductivity in relation to the stem mechanical strength of Paeonia lactiflora.

The quality of cut Paeonia lactiflora flowers is limited by their low stem mechanical strength, but the underlying mechanism of this low strength is poorly understood. In this study, two P. lactiflora cultivars with distinct stem mechanical strengths (Chui Touhong with low stem mechanical strength and Da Fugui with high stem mechanical strength) were used as test materials. The xylem development was examined at the cellular level, and the phloem conductivity was analyzed by evaluating phloem geometry. The results showed that the secondary cell wall formation of the xylem of Chui Touhong was affected primarily in fiber cells but was affected little in vessel cells. The formation of the secondary cell walls in the xylem fiber cells of Chui Touhong was delayed, resulting in longer and thinner fiber cells with a lack of cellulose and S-lignin in the secondary cell walls. Moreover, the phloem conductivity of Chui Touhong was lower than that of Da Fugui, and more callose was accumulated in the lateral walls of the phloem sieve elements of Chui Touhong. Consequently, the delayed deposition of the secondary cell walls of the xylem fiber cells was the main factor leading to the low stem mechanical strength of Chui Touhong, and the low stem mechanical strength was closely related to the low conductivity of sieve tubes and extensive callose accumulation in the phloem. These findings provide a new perspective on enhancing P. lactiflora stem mechanical strength by targeting single cell level, and lay the foundation for future works on the correlation between phloem long-distance transport and stem mechanical strength.

Shade effects on growth, photosynthesis and chlorophyll fluorescence parameters of three Paeonia species.

Insufficient light intensity inhibits the growth of cultivated herbaceous peony and decreases its economic value. Owing to the increased demand for shade-tolerant herbaceous peony, the selection of appropriate parents for hybridization is essential. Paeonia anomala, Paeonia intermedia and Paeonia veitchii can grow under shade conditions in their natural habitats; however, their photosynthetic capacities under shade have not been studied. In this study, we simulated low light intensity (30% sunlight) and evaluated the morphological, photosynthetic and chlorophyll fluorescence parameters of these three species. Moreover, the shade tolerance of these species as well as two common cultivars (Paeonia lactiflora 'Da Fugui', which is suitable for solar greenhouse cultivation, and P. lactiflora 'Qiao Ling', which is not suitable for solar greenhouse cultivation) was evaluated. The results showed that under shade, the leaf area of P. anomala and P. intermedia increased, the single flowering period of P. intermedia and P. veitchii was prolonged, and the flower color of P. veitchii faded. With respect to P. anomala, P. intermedia and P. veitchii, shade eliminated the photosynthetic 'lunch break' phenomenon and decreased photoinhibition at midday. Furthermore, the maximum photochemical efficiency (Fv/Fm) and maximum primary photochemical yield (Fv/Fo) of photosystem II (PSII) in the three species improved significantly, and their changes in light dissipation were different. The shade tolerance of the tested accessions was in the order P. veitchii > P. intermedia > P. anomala > 'Da Fugui' > 'Qiao Ling', showing that the three wild species were better adapted to low light intensity than the cultivars. Thus, P. anomala, P. intermedia and P. veitchii could potentially be used in the development of shade-tolerant herbaceous peony cultivars.

Characteristics of Microsatellites Mined from Transcriptome Data and the Development of Novel Markers in Paeonia lactiflora.

The insufficient number of available simple sequence repeats (SSRs) inhibits genetic research on and molecular breeding of Paeonia lactiflora, a flowering crop with great economic value. The objective of this study was to develop SSRs for P. lactiflora with Illumina RNA sequencing and assess the role of SSRs in gene regulation. The results showed that dinucleotides with AG/CT repeats were the most abundant type of repeat motif in P. lactiflora and were preferentially distributed in untranslated regions. Significant differences in SSR size were observed among motif types and locations. A large number of unigenes containing SSRs participated in catalytic activity, metabolic processes and cellular processes, and 28.16% of all transcription factors and 21.74% of hub genes for inflorescence stem straightness were found to contain SSRs. Successful amplification was achieved with 89.05% of 960 pairs of SSR primers, 55.83% of which were polymorphic, and most of the 46 tested primers had a high level of transferability to the genus Paeonia. Principal component and cluster dendrogram analyses produced results consistent with known genealogical relationships. This study provides a set of SSRs with abundant information for future accession identification, marker-trait association and molecular assisted breeding in P. lactiflora.

Transcriptome and weighted correlation network analyses provide insights into inflorescence stem straightness in Paeonia lactiflora.

KEY MESSAGE: Lack of structural components results in inflorescence stem bending. Differentially expressed genes involved in lignin and hemicellulose biosynthesis are vital; genes involved in cellulose and glycan biosynthesis are also relevant. An erect inflorescence stem is essential for high-quality cut herbaceous peony flowers. To explore the factors underlying inflorescence stem bending, major cell walls contents were measured, and stem structure was observed in two herbaceous peony varieties with contrasting stem straightness traits ('Da Fugui', upright; 'Chui Touhong', bending). In addition, Illumina sequencing was performed and weighted correlation network analysis (WGCNA) was used to analyze the results. The results showed significant differences in lignin, hemicellulose and soluble sugar contents, sclerenchyma and xylem areas and thickening in cell walls in pith at stage S3, when bending begins. In addition, 44,182 significantly differentially expressed genes (DEGs) were found, and these DEGs were mainly enriched in 36 pathways. Among the DEGs, hub genes involved in lignin, cellulose, and xylan biosynthesis and transcription factors that regulated these process were identified by WGCNA. These results suggested that the contents of compounds that provided cell wall rigidity were vital factors affecting inflorescence stem straightness in herbaceous peony. Genes involved in or regulating the biosynthesis of these compounds are thus important; lignin and hemicellulose are of great interest, and cellulose and glycan should not be ignored. This paper lays a foundation for developing new herbaceous peony varieties suitable for cut flowers by molecular-assisted breeding.

Selection and validation of reference genes of Paeonia lactiflora in growth development and light stress.

The stem of Paeonia lactiflora will bend when it grows in greenhouse at a low light intensity. It is important to explore causes of morphological changes of peony to improve its quality. Gene expression can be evaluated by quantitative real-time PCR, based on reference gene. However, systematic selection of reference genes under weak lighting for herbaceous peony is lacking. To address this problem, we first selected 10 candidate reference genes based on a coefficient of variation of gene expression from peony stem transcriptome data. Then, geNorm, NormFinder and BestKeeper were applied to assess the stability of the genes, and RankAggreg was used to give a comprehensive ranking. The results show that there are some differences in optimal reference genes among samples from different organs and under the two lighting conditions, and the optimal number of suitable reference genes is distinct. Two selected suitable reference genes were then used to normalize target genes, and the results were compared with transcriptome data. Consistent gene expression trends were obtained, indicating the reliability of the method. To the best of our knowledge, this is the first time reference genes for herbaceous peony were selected in different organs, developmental stages and under two kinds of lighting conditions. The findings can provide a practical method for selecting reference genes for peony under these conditions and demonstrate a useful combination of reference genes.