A Combined Metabolome and Transcriptome Reveals the Lignin Metabolic Pathway during the Developmental Stages of Peel Coloration in the 'Xinyu' Pear.

Sand pear is the main cultivated pear species in China, and brown peel is a unique feature of sand pear. The formation of brown peel is related to the activity of the cork layer, of which lignin is an important component. The formation of brown peel is intimately associated with the biosynthesis and accumulation of lignin; however, the regulatory mechanism of lignin biosynthesis in pear peel remains unclear. In this study, we used a newly bred sand pear cultivar 'Xinyu' as the material to investigate the biosynthesis and accumulation of lignin at nine developmental stages using metabolomic and transcriptomic methods. Our results showed that the 30 days after flowering (DAF) to 50DAF were the key periods of lignin accumulation according to data analysis from the assays of lignin measurement, scanning electron microscope (SEM) observation, metabolomics, and transcriptomics. Through weighted gene co-expression network analysis (WGCNA), positively correlated modules with lignin were identified. A total of nine difference lignin components were identified and 148 differentially expressed genes (DEGs), including 10 structural genes (PAL1, C4H, two 4CL genes, HCT, CSE, two COMT genes, and two CCR genes) and MYB, NAC, ERF, and TCP transcription factor genes were involved in lignin metabolism. An analysis of RT-qPCR confirmed that these DEGs were involved in the biosynthesis and regulation of lignin. These findings further help us understand the mechanisms of lignin biosynthesis and provide a theoretical basis for peel color control and quality improvement in pear breeding and cultivation.

Integrated Methylome and Transcriptome Analysis between Wizened and Normal Flower Buds in Pyrus pyrifolia Cultivar 'Sucui 1'.

Here, cytosine methylation in the whole genome of pear flower buds was mapped at a single-base resolution. There was 19.4% methylation across all sequenced C sites in the Pyrus pyrifolia cultivar 'Sucui 1' flower bud genome. Meantime, the CG, CHG, and CHH sequence contexts (where H = A, T or C) exhibited 47.4%, 33.3%, and 11.9% methylation, respectively. Methylation in different gene regions was revealed through combining methylome and transcriptome analysis, which presented various transcription trends. Genes with methylated promoters exhibited lower expression levels than genes with non-methylated promoters, while body-methylated genes displayed an obvious negative correlation with their transcription levels. The methylation profiles of auxin- and cytokinin-related genes were estimated. And some of them proved to be hypomethylated, with increased transcription levels, in wizened buds. More specifically, the expression of the genes PRXP73, CYP749A22, and CYP82A3 was upregulated as a result of methylation changes in their promoters. Finally, auxin and cytokinin concentrations were higher in wizened flower buds than in normal buds. The exogenous application of paclobutrazol (PP333) in the field influenced the DNA methylation status of some genes and changed their expression level, reducing the proportion of wizened flower buds in a concentration-dependent manner. Overall, our results demonstrated the relationship between DNA methylation and gene expression in wizened flower buds of P. pyrifolia cultivar 'Sucui 1', which was associated with changes in auxin and cytokinin concentrations.

Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

Allelic variation of BBX24 is a dominant determinant controlling red coloration and dwarfism in pear.

Variation in anthocyanin biosynthesis in pear fruit provides genetic germplasm resources for breeding, while dwarfing is an important agronomic trait, which is beneficial to reduce the management costs and allow for the implementation of high-density cultivation. Here, we combined bulked segregant analysis (BSA), quantitative trait loci (QTL), and structural variation (SV) analysis to identify a 14-bp deletion which caused a frame shift mutation and resulted in the premature translation termination of a B-box (BBX) family of zinc transcription factor, PyBBX24, and its allelic variation termed PyBBX24ΔN14. PyBBX24ΔN14 overexpression promotes anthocyanin biosynthesis in pear, strawberry, Arabidopsis, tobacco, and tomato, while that of PyBBX24 did not. PyBBX24ΔN14 directly activates the transcription of PyUFGT and PyMYB10 through interaction with PyHY5. Moreover, stable overexpression of PyBBX24ΔN14 exhibits a dwarfing phenotype in Arabidopsis, tobacco, and tomato plants. PyBBX24ΔN14 can activate the expression of PyGA2ox8 via directly binding to its promoter, thereby deactivating bioactive GAs and reducing the plant height. However, the nuclear localization signal (NLS) and Valine-Proline (VP) motifs in the C-terminus of PyBBX24 reverse these effects. Interestingly, mutations leading to premature termination of PyBBX24 were also identified in red sports of un-related European pear varieties. We conclude that mutations in PyBBX24 gene link both an increase in pigmentation and a decrease in plant height.

Genome-wide analysis of the CCT gene family in Chinese white pear (Pyrus bretschneideri Rehd.) and characterization of PbPRR2 in response to varying light signals.

BACKGROUND: Canopy architecture is critical in determining the light environment and subsequently the photosynthetic productivity of fruit crops. Numerous CCT domain-containing genes are crucial for plant adaptive responses to diverse environmental cues. Two CCT genes, the orthologues of AtPRR5 in pear, have been reported to be strongly correlated with photosynthetic performance under distinct canopy microclimates. However, knowledge concerning the specific expression patterns and roles of pear CCT family genes (PbCCTs) remains very limited. The key roles played by PbCCTs in the light response led us to examine this large gene family in more detail. RESULTS: Genome-wide sequence analysis identified 42 putative PbCCTs in the genome of pear (Pyrus bretschneideri Rehd.). Phylogenetic analysis indicated that these genes were divided into five subfamilies, namely, COL (14 members), PRR (8 members), ZIM (6 members), TCR1 (6 members) and ASML2 (8 members). Analysis of exon-intron structures and conserved domains provided support for the classification. Genome duplication analysis indicated that whole-genome duplication/segmental duplication events played a crucial role in the expansion of the CCT family in pear and that the CCT family evolved under the effect of purifying selection. Expression profiles exhibited diverse expression patterns of PbCCTs in various tissues and in response to varying light signals. Additionally, transient overexpression of PbPRR2 in tobacco leaves resulted in inhibition of photosynthetic performance, suggesting its possible involvement in the repression of photosynthesis. CONCLUSIONS: This study provides a comprehensive analysis of the CCT gene family in pear and will facilitate further functional investigations of PbCCTs to uncover their biological roles in the light response.

A mutation in the promoter of the arabinogalactan protein 7-like gene PcAGP7-1 affects cell morphogenesis and brassinolide content in pear (Pyrus communis L.) stems.

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.

Comprehensive Comparative Analysis of the GATA Transcription Factors in Four Rosaceae Species and Phytohormonal Response in Chinese Pear (Pyrus bretschneideri) Fruit.

The GATA gene family is one of the most important transcription factors (TFs). It extensively exists in plants, contributes to diverse biological processes such as the development process, and responds to environmental stress. Although the GATA gene family has been comprehensively and systematically studied in many species, less is known about GATA genes in Chinese pears (Pyrus bretschneideri). In the current study, the GATA gene family in the four Rosaceae genomes was identified, its structural characteristics identified, and a comparative analysis of its properties was carried out. Ninety-two encoded GATA proteins were authenticated in the four Rosaceae genomes (Pyrus bretschneideri, Prunus avium, Prunus mume, and Prunus persica) and categorized into four subfamilies (Ⅰ-Ⅳ) according to phylogeny. The majority of GATA genes contained one to two introns and conserved motif composition analysis revealed their functional divergence. Whole-genome duplications (WGDs) and dispersed duplication (DSD) played a key role in the expansion of the GATA gene family. The microarray indicated that, among P. bretschneideri, P. avium, P. mume and P. persica, GATA duplicated regions were more conserved between Pyrus bretschneideri and Prunus persica with 32 orthologous genes pairs. The physicochemical parameters, duplication patterns, non-synonymous (ka), and synonymous mutation rate (ks) and GO annotation ontology were performed using different bioinformatics tools. cis-elements respond to various phytohormones, abiotic/biotic stress, and light-responsive were found in the promoter regions of GATA genes which were induced via stimuli. Furthermore, subcellular localization of the PbGATA22 gene product was investigated, showing that it was present in the nucleus of tobacco (Nicotiana tabacum) epidermal cells. Finally, in silico analysis was performed on various organs (bud, leaf, stem, ovary, petal, and sepal) and different developmental stages of fruit. Subsequently, the expression profiles of PbGATA genes were extensively expressed under exogenous hormonal treatments of SA (salicylic acid), MeJA (methyl jasmonate), and ABA (abscisic acid) indicating that play important role in hormone signaling pathways. A comprehensive analysis of GATA transcription factors was performed through systematic biological approaches and comparative genomics to establish a theoretical base for further structural and functional investigations in Rosaceae species.

Pear xyloglucan endotransglucosylase/hydrolases PcBRU1 promotes stem growth through regulating cell wall elongation.

Brassinosteroids (BRs) play numerous important roles in plant growth and development. Previous studies reported that BRs could promote stem growth by regulating the expression of xyloglucan endotransglucosylase/hydrolases (XTHs). However, the mechanism of XTHs involved in stem growth remains unclear. In this study, PcBRU1, which belonged to the XTH family, was upregulated by exogenous BL treatment in Pyrus communis. The expression of PcBRU1 was highest in stems and lowest in leaves. Subcellular localization analysis indicated that PcBRU1 was located in the plasma membrane. Furthermore, overexpressing PcBRU1 in tobaccos promoted the plant height and internode length. Electron microscopy and anatomical structure analysis showed that the cell wall was significantly thinner and the cells were slenderer in transgenic tobacco lines overexpressing PcBRU1 than in wild-type tobaccos. PcBRU1 promoted stem growth as it loosened the cell wall, leading to the change in cell morphology. In addition, overexpressing PcBRU1 altered the root development and leaf shape of transgenic tobaccos. Taken together, the results could provide a theoretical basis for the XTH family in regulating cell-wall elongation and stem growth.

Florigen governs shoot regeneration.

It is widely known that during the reproductive stage (flowering), plants do not root well. Most protocols of shoot regeneration in plants utilize juvenile tissue. Adding these two realities together encouraged us to study the role of florigen in shoot regeneration. Mature tobacco tissue that expresses the endogenous tobacco florigen mRNA regenerates poorly, while juvenile tissue that does not express the florigen regenerates shoots well. Inhibition of Nitric Oxide (NO) synthesis reduced shoot regeneration as well as promoted flowering and increased tobacco florigen level. In contrast, the addition of NO (by way of NO donor) to the tissue increased regeneration, delayed flowering, reduced tobacco florigen mRNA. Ectopic expression of florigen genes in tobacco or tomato decreased regeneration capacity significantly. Overexpression pear PcFT2 gene increased regeneration capacity. During regeneration, florigen mRNA was not changed. We conclude that florigen presence in mature tobacco leaves reduces roots and shoots regeneration and is the possible reason for the age-related decrease in regeneration capacity.

A Bifunctional Peptide Conjugate That Controls Infections of Erwinia amylovora in Pear Plants.

In this paper, peptide conjugates were designed and synthesized by incorporating the antimicrobial undecapeptide BP16 at the C- or N-terminus of the plant defense elicitor peptide flg15, leading to BP358 and BP359, respectively. The evaluation of their in vitro activity against six plant pathogenic bacteria revealed that BP358 displayed MIC values between 1.6 and 12.5 µM, being more active than flg15, BP16, BP359, and an equimolar mixture of BP16 and flg15. Moreover, BP358 was neither hemolytic nor toxic to tobacco leaves. BP358 triggered the overexpression of 6 out of the 11 plant defense-related genes tested. Interestingly, BP358 inhibited Erwinia amylovora infections in pear plants, showing slightly higher efficacy than the mixture of BP16 and flg15, and both treatments were as effective as the antibiotic kasugamycin. Thus, the bifunctional peptide conjugate BP358 is a promising agent to control fire blight and possibly other plant bacterial diseases.

Downstream of GA4, PbCYP78A6 participates in regulating cell cycle-related genes and parthenogenesis in pear (Pyrus bretshneideri Rehd.).

BACKGROUND: Parthenocarpy results in traits attractive to both consumers and breeders, and it overcomes the obstacle of self-incompatibility in the fruit set of horticultural crops, including pear (Pyrus bretshneider). However, there is limited knowledge regarding the genetic and molecular mechanisms that regulate parthenogenesis. RESULTS: Here, in a transcriptional comparison between pollination-dependent fruit and GA4-induced parthenocarpy, PbCYP78A6 was identified and proposed as a candidate gene involved in parthenocarpy. PbCYP78A6 is similar to Arabidopsis thaliana CYP78A6 and highly expressed in pear hypanthia. The increased PbCYP78A6 expression, as assessed by RT-qPCR, was induced by pollination and GA4 exposure. The ectopic overexpression of PbCYP78A6 contributed to parthenocarpic fruit production in tomato. The PbCYP78A6 expression coincided with fertilized and parthenocarpic fruitlets development and the expression of fruit development-related genes as assessed by cytological observations and RT-qPCR, respectively. PbCYP78A6 RNA interference and overexpression in pear calli revealed that the gene is an upstream regulator of specific fruit development-related genes in pear. CONCLUSIONS: Our findings indicate that PbCYP78A6 plays a critical role in fruit formation and provide insights into controlling parthenocarpy.

SMRT sequencing of full-length transcriptome of birch-leaf pear (Pyrus betulifolia Bunge) under drought stress.

Drought limits the pear yield and quality. The birch-leaf pear (Pyrus betulifolia Bunge) is one of the most frequently used pear rootstocks. Identifying genes involved in drought resistance of P. betulifolia would suggest candidate genes for molecular breeding. We used single-molecule long-read sequencing technology to investigate the transcriptome of birch-leaf pear under drought stress. As a result, 362,139 consensus reads were identified using six databases, among which 342,162 genes were functionally annotated. Further, we identified 7094 long noncoding RNAs. The sequencing data contained 9891 alternative splicing and 100,836 alternative polyadenylation events. We report here the full-length sequence of birch-leaf pear, which can be used for breeding enhanced varieties.

PbSRT1 and PbSRT2 regulate pear growth and ripening yet displaying a species-specific regulation in comparison to other Rosaceae spp.

Epigenetic regulation is crucial to ensure a coordinated control of the different events that occur during fruit development and ripening. Sirtuins are NAD+-dependent histone deacetylases involved in the regulation of gene expression of many biological processes. However, their implications in the Rosaceae family remains unexplored. Accordingly, in this work, we demonstrated the phylogenetic divergence of both sirtuins among Rosaceae species. We then characterized the expression pattern of both SRT1 and SRT2 in selected pome and stone fruit species. Both SRT1 and SRT2 significantly changed during the fruit development and ripening of apple, nectarine and pear fruit, displaying a different expression profile. Such differences could explain in part their different ripening behaviour. To further unravel the role of sirtuins on the fruit development and ripening processes, a deeper analysis was performed using pear as a fruit model. In pear, PbSRT1 gene expression levels were negatively correlated with specific hormones (i.e. abscisic acid, indole-3-acetic acid, gibberellin A1 and zeatin) during the first phases of fruit development. PbSRT2 seemed to directly mediate pear ripening in an ethylene-independent manner. This hypothesis was further reinforced by treating the fruit with the ethylene inhibitor 1-methylcyclopropene (1-MCP). Instead, enhanced PbSRT2 along pear growth/ripening positively correlated with the accumulation of major sugars (R2 > 0.94), reinforcing the idea that sugar metabolism may be a target of epigenetic modifications during fruit ripening. Overall, the results from this study point out, for the first time, the importance that sirtuins have in the regulation of fruit growth and ripening of pear fruit by likely regulating hormonal and sugar metabolism.

PbCSE1 promotes lignification during stone cell development in pear (Pyrus bretschneideri) fruit.

Pear [Pyrus bretschneideri cv. Dangshan Su] fruit quality is not always satisfactory owing to the presence of stone cells, and lignin is the main component of stone cells in pear fruits. Caffeoyl shikimate esterase (CSE) is a key enzyme in the lignin biosynthesis. Although CSE-like genes have been isolated from a variety of plant species, their orthologs are not characterized in pear. In this study, the CSE gene family (PbCSE) from P. bretschneideri was identified. According to the physiological data and quantitative RT-PCR (qRT-PCR), PbCSE1 was associated with lignin deposition and stone cell formation. The overexpression of PbCSE1 increased the lignin content in pear fruits. Relative to wild-type (WT) Arabidopsis, the overexpression of PbCSE1 delayed growth, increased the lignin deposition and lignin content in stems. Simultaneously, the expression of lignin biosynthetic genes were also increased in pear fruits and Arabidopsis. These results demonstrated that PbCSE1 plays an important role in cell lignification and will provide a potential molecular strategy to improve the quality of pear fruits.

The mechanism for brassinosteroids suppressing climacteric fruit ripening.

The plant hormone ethylene is important for the ripening of climacteric fruit, such as pear (Pyrus ussuriensis), and the brassinosteroid (BR) class of phytohormones affects ethylene biosynthesis during ripening via an unknown molecular mechanism. Here, we observed that exogenous BR treatment suppressed ethylene production and delayed fruit ripening, whereas treatment with a BR biosynthesis inhibitor promoted ethylene production and accelerated fruit ripening in pear, suggesting BR is a ripening suppressor. The expression of the transcription factor BRASSINAZOLE-RESISTANT 1PuBZR1 was enhanced by BR treatment during pear fruit ripening. PuBZR1 interacted with PuACO1, which converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, and suppressed its activity. BR-activated PuBZR1 bound to the promoters of PuACO1 and of PuACS1a, which encodes ACC synthase, and directly suppressed their transcription. Moreover, PuBZR1 suppressed the expression of transcription factor PuERF2 by binding its promoter, and PuERF2 bound to the promoters of PuACO1 and PuACS1a. We concluded that PuBZR1 indirectly suppresses the transcription of PuACO1 and PuACS1a through its regulation of PuERF2. Ethylene production and expression profiles of corresponding apple (Malus domestica) homologs showed similar changes following epibrassinolide treatment. Together, these results suggest that BR-activated BZR1 suppresses ACO1 activity and the expression of ACO1 and ACS1, thereby reducing ethylene production and suppressing fruit ripening. This likely represents a conserved mechanism by which BR suppresses ethylene biosynthesis during climacteric fruit ripening.

Linking wood anatomy with growth vigour and susceptibility to alternate bearing in composite apple and pear trees.

Excess vegetative growth and irregular fruit-bearing are often undesirable in horticultural practice. However, the biological mechanisms underlying these traits in fruit trees are not fully understood. Here, we tested if growth vigour and susceptibility of apple and pear trees to alternate fruit-bearing are associated with vascular anatomy. We examined anatomical traits related to water transport and nutrient storage in young woody shoots and roots of 15 different scion/rootstock cultivars of apple and pear trees. In addition, soil and leaf water potentials were measured across a drought period. We found a positive correlation between the mean vessel diameter of roots and the annual shoot length. Vigorously growing trees also maintained less negative midday leaf water potential during drought. Furthermore, we observed a close negative correlation between the proportions of total parenchyma in the shoots and the alternate bearing index. Based on anatomical proxies, our results suggest that xylem transport efficiency of rootstocks is linked to growth vigour of both apple and pear trees, while limited carbohydrate storage capacity of scions may be associated with increased susceptibility to alternate bearing. These findings can be useful for the breeding of new cultivars of commercially important fruit trees.

Metabolomic and transcriptomic analyses highlight the influence of lipid changes on the post-harvest softening of Pyrus ussurian Max. 'Zaoshu Shanli'.

How lipids influence post-harvest softening in pears is not well understood. LC-MS/MS (Liquid chromatography-tandem mass spectrometry) and RNA-Seq analyses of 'Zaoshu Shanli' (ZSSL) pears were conducted during post-harvest storage. This approach enabled the identification of 98 different metabolites that upregulated and 95 that downregulated at 18 days post-harvest in ZSSL fruits to day 0. Metabolites were significantly enriched in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways including glycerophospholipid metabolism and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. When comparing fruits from day 18 to those from day 0 post-harvest, RNA-seq analyses further highlighted 6496 differentially expressed genes (DEGs) in ZSSL fruits that were significantly enriched in KEGG pathways including glycerophospholipid metabolism and fatty acid degradation. Overall, these results suggested that glycerophospholipid metabolism is closely related to the post-harvest softening of pears. Further research will be essential in order to fully explore the functional implications of and mechanistic basis for these findings.

Effect of 1-MCP and ethylene absorbent on the development of lenticel disorder of 'Xinli No.7' pear and possible mechanisms.

BACKGROUD: A common lenticel disorder which occurs in the peel of 'Xinli No. 7' pears (Pyrus bretschneideri Rehd.) had not previously been described. Symptoms of this lenticel disorder include enlarging and bulging of the lenticels which results in significant commercial losses. Understanding the physiological basis of lenticel disorder and developing practical methods to control it is crucial for the successful marketing of this pear. RESULTS: The development of this lenticel disorder was found to be closely related to the endogenous ethylene production during storage. 1-Methylcyclopropene (1-MCP) combined with an ethylene absorbent (EA) treatment was found to significantly reduce the development of the disorder by inhibiting the expression of ethylene related genes, PbACS1, PbACS2 and PbACO. It is proposed that the enlarged lenticels may result from increased lignin accumulation in the peel cells, which is inhibited by this combined postharvest treatment. It was shown that the expression of six lignin related genes decreased following the treatment. The results suggest that PbPAL, Pb4CL and PbCAD could be critical in regulating the development of this lenticel disorder. CONCLUSION: Endogenous ethylene plays a key role in the development of this lenticel disorder in 'Xinli No. 7' pear. The enlarged lenticels which is characteristic of this disorder maybe related to increased lignin accumulation in the peel cells, which were inhibited with 1-MCP combined with an EA treatment. These results provide a practical method for managing the development of lenticel disorder in 'Xinli No. 7' pear and helps clarify the developmental mechanisms of this disorder. © 2020 Society of Chemical Industry.

High throughput mathematical modeling and multi-objective evolutionary algorithms for plant tissue culture media formulation: Case study of pear rootstocks.

Simplified prediction of the interactions of plant tissue culture media components is of critical importance to efficient development and optimization of new media. We applied two algorithms, gene expression programming (GEP) and M5' model tree, to predict the effects of media components on in vitro proliferation rate (PR), shoot length (SL), shoot tip necrosis (STN), vitrification (Vitri) and quality index (QI) in pear rootstocks (Pyrodwarf and OHF 69). In order to optimize the selected prediction models, as well as achieving a precise multi-optimization method, multi-objective evolutionary optimization algorithms using genetic algorithm (GA) and particle swarm optimization (PSO) techniques were compared to the mono-objective GA optimization technique. A Gamma test (GT) was used to find the most important determinant input for optimizing each output factor. GEP had a higher prediction accuracy than M5' model tree. GT results showed that BA (Γ = 4.0178), Mesos (Γ = 0.5482), Mesos (Γ = 184.0100), Micros (Γ = 136.6100) and Mesos (Γ = 1.1146), for PR, SL, STN, Vitri and QI respectively, were the most important factors in culturing OHF 69, while for Pyrodwarf culture, BA (Γ = 10.2920), Micros (Γ = 0.7874), NH4NO3 (Γ = 166.410), KNO3 (Γ = 168.4400), and Mesos (Γ = 1.4860) were the most important influences on PR, SL, STN, Vitri and QI respectively. The PSO optimized GEP models produced the best outputs for both rootstocks.

Transcriptome Analysis of Pyrus betulaefolia Seedling Root Responses to Short-Term Potassium Deficiency.

Potassium (K) plays a crucial role in multiple physiological and developmental processes in plants. Its deficiency is a common abiotic stress that inhibits plant growth and reduces crop productivity. A better understanding of the mechanisms involved in plant responses to low K could help to improve the efficiency of K use in plants. However, such responses remain poorly characterized in fruit tree species such as pears (Pyrus sp). We analyzed the physiological and transcriptome responses of a commonly used pear rootstock, Pyrus betulaefolia, to K-deficiency stress (0 mM). Potassium deprivation resulted in apparent changes in root morphology, with short-term low-K stress resulting in rapidly enhanced root growth. Transcriptome analyses indicated that the root transcriptome was coordinately altered within 6 h after K deprivation, a process that continued until 15 d after treatment. Potassium deprivation resulted in the enhanced expression (up to 5-fold) of a putative high-affinity K+ transporter, PbHAK5 (Pbr037826.1), suggesting the up-regulation of mechanisms associated with K+ acquisition. The enhanced root growth in response to K-deficiency stress was associated with a rapid and sustained decrease in the expression of a transcription factor, PbMYB44 (Pbr015309.1), potentially involved in mediating auxin responses, and the increased expression of multiple genes associated with regulating root growth. The concentrations of several phytohormones including indoleacetic acid (IAA), ABA, ETH, gibberellin (GA3), and jasmonic acid (JA) were higher in response to K deprivation. Furthermore, genes coding for enzymes associated with carbon metabolism such as SORBITOL DEHYDROGENASE (SDH) and SUCROSE SYNTHASE (SUS) displayed greatly enhanced expression in the roots under K deprivation, presumably indicating enhanced metabolism to meet the increased energy demands for growth and K+ acquisition. Together, these data suggest that K deprivation in P. betulaefolia results in the rapid re-programming of the transcriptome to enhance root growth and K+ acquisition. These data provide key insights into the molecular basis for understanding low-K-tolerance mechanisms in pears and in other related fruit trees and identifying potential candidates that warrant further analyses.