GWAS identifies two important genes involved in Chinese chestnut weight and leaf length regulation.

There are many factors that affect the yield of Chinese chestnut (Castanea mollissima), with single nut weight (SNW) being one of the most important. Leaf length is also related to Chinese chestnut yield. However, the genetic architecture and gene function associated with Chinese chestnut nut yield have not been fully explored. In this study, we performed genotyping by sequencing 151 Chinese chestnut cultivars, followed by a genome-wide association study (GWAS) on six horticultural traits. First, we analyzed the phylogeny of the Chinese chestnut and found that the Chinese chestnut cultivars divided into two ecotypes, a northern and southern cultivar group. Differences between the cultivated populations were found in the pathways of plant growth and adaptation to the environment. In the selected regions, we also found interesting tandemly arrayed genes that may influence Chinese chestnut traits and environmental adaptability. To further investigate which horticultural traits were selected, we performed a GWAS using six horticultural traits from 151 cultivars. Forty-five loci that strongly associated with horticultural traits were identified, and six genes highly associated with these traits were screened. In addition, a candidate gene associated with SNW, APETALA2 (CmAP2), and another candidate gene associated with leaf length (LL), CRYPTOCHROME INTERACTING BASIC HELIX-LOOP-HELIX 1 (CmCIB1), were verified in Chinese chestnut and Arabidopsis (Arabidopsis thaliana). Our results showed that CmAP2 affected SNW by negatively regulating cell size. CmCIB1 regulated the elongation of new shoots and leaves by inducing cell elongation, potentially affecting photosynthesis. This study provided valuable information and insights for Chinese chestnut breeding research.

Changes of cell wall components during embryogenesis of Castanea mollissima.

The Chinese chestnut (Castanea mollissima Blume) 'Huaihuang' was chosen as the experimental material to observe embryogenesis and the dynamic changes of cell wall components during this process. Various developmental stages of embryos, including globular embryos, heart embryos, torpedo embryos and cotyledon embryos, were observed. The results showed that during embryogenesis, cellulose increased, and callose rapidly degraded. In the cell walls of developing embryos, pectic homogalacturonan (HG), especially low-esterified HG, was abundant, suggesting rapid synthesis and de-methyl-esterification of HG. Extensin and galactan increased with the development of the embryos. In contrast, the arabinan epitopes decreased in developing embryos but were more abundant than galactan epitopes at all stages. Xylan epitopes showed explicit boundaries between the outer epidermal wall and the rest of the inner tissues, and the fluorescence intensity of the outer epidermal wall was significantly higher than that of the inner tissues. Furthermore, the results indicated that the outer epidermal wall contained high amounts of cellulose, HG pectin and hemicellulose, especially arabinan and xylan. These results suggested the presence of rapid pectin metabolism, cellulose synthesis, rapid degradation of callose, different distributive patterns and dynamic changes of hemicellulose (galactan, arabinan and xylan) and extensin during embryogenesis. Various cell wall components exist in different tissues of the embryo, and dynamic changes in cell wall components are involved in the embryonic development process.

Roles of the GA-mediated SPL Gene Family and miR156 in the Floral Development of Chinese Chestnut (Castanea mollissima).

Chestnut (Castanea mollissima) is a deciduous tree species with major economic and ecological value that is widely used in the study of floral development in woody plants due its monoecious and out-of-proportion characteristics. Squamosa promoter-binding protein-like (SPL) is a plant-specific transcription factor that plays an important role in floral development. In this study, a total of 18 SPL genes were identified in the chestnut genome, of which 10 SPL genes have complementary regions of CmmiR156. An analysis of the phylogenetic tree of the squamosa promoter-binding protein (SBP) domains of the SPL genes of Arabidopsis thaliana, Populus trichocarpa, and C. mollissima divided these SPL genes into eight groups. The evolutionary relationship between poplar and chestnut in the same group was similar. A structural analysis of the protein-coding regions (CDSs) showed that the domains have the main function of SBP domains and that other domains also play an important role in determining gene function. The expression patterns of CmmiR156 and CmSPLs in different floral organs of chestnut were analyzed by real-time quantitative PCR. Some CmSPLs with similar structural patterns showed similar expression patterns, indicating that the gene structures determine the synergy of the gene functions. The application of gibberellin (GA) and its inhibitor (Paclobutrazol, PP333) to chestnut trees revealed that these exert a significant effect on the number and length of the male and female chestnut flowers. GA treatment significantly increased CmmiR156 expression and thus significantly decreased the expression of its target gene, CmSPL6/CmSPL9/CmSPL16, during floral bud development. This finding indicates that GA might indirectly affect the expression of some of the SPL target genes through miR156. In addition, RNA ligase-mediated rapid amplification of the 5' cDNA ends (RLM-RACE) experiments revealed that CmmiR156 cleaves CmSPL9 and CmSPL16 at the 10th and 12th bases of the complementary region. These results laid an important foundation for further study of the biological function of CmSPLs in the floral development of C. mollissima.

New insights into the evolution and local adaptation of the genus Castanea in east Asia.

Chestnut plants (Castanea) are important nut fruit trees worldwide. However, little is known regarding the genetic relationship and evolutionary history of different species within the genus. How modern chestnut plants have developed local adaptation to various climates remains a mystery. The genomic data showed that Castanea henryi first diverged in the Oligocene ~31.56 million years ago, followed by Castanea mollissima, and the divergence between Castanea seguinii and Castanea crenata occurred in the mid-Miocene. Over the last 5 million years, the population of chestnut plants has continued to decline. A combination of selective sweep and environmental association studies was applied to investigate the genomic basis of chestnut adaptation to different climates. Twenty-two candidate genes were associated with temperature and precipitation. We also revealed the molecular mechanism by which CmTOE1 interacts with CmZFP8 and CmGIS3 to promote the formation of non-glandular trichomes for adaptation to low temperature and high altitudes. We found a significant expansion of CER1 genes in Chinese chestnut (C. mollissima) and verified the CmERF48 regulation of CmCER1.6 adaptation to drought environments. These results shed light on the East Asian chestnut plants as a monophyletic group that had completed interspecific differentiation in the Miocene, and provided candidate genes for future studies on adaptation to climate change in nut trees.

Low Concentration of Aluminum-Stimulated Pollen Tube Growth of Apples (Malus domestica).

Aluminum (Al) is an important element in soil constitution. Previous studies have shown that high concentration of Al affects the normal growth of crops, resulting in crop yield reduction and inferior quality. Nevertheless, Al has also been referred to as a beneficial element, especially when used at low concentrations, but the cytological mechanism is not clear. Influences of low concentration AlCl3 on the pollen tube growth of apple (Malus domestica) and its possible cytological mechanism were investigated in this study. The results showed that 20 µM AlCl3 promoted pollen germination and tube elongation; 20 µM AlCl3 enhanced Ca2+ influx but did not affect [Ca2+]c of the pollen tube tip; and 20 µM AlCl3 decreased acid pectins in pollen tubes but increased esterified pectins and arabinan pectins in pollen tubes. According to the information provided in this research, 20 µM AlCl3 stimulated growth of pollen tubes by enhancing Ca2+ influx and changing cell wall components.

Effect of boron toxicity on pollen tube cell wall architecture and the relationship of cell wall components of Castanea mollissima Blume.

Boron (B) is essential to plant development. However, excessive B is toxic to plants. This research was performed to evaluate the effects of B toxicity on cell wall architecture of Chinese chestnut (Castanea mollissima Blume) pollen tubes with emphasis on the relationship among pectins, cellulose, and callose. Results showed that 0.8 mM H3BO3 inhibited pollen germination and led to abnormal morphology of the pollen tubes. B toxicity also affected the distribution of cell wall components of the pollen tube. In control pollen tube, esterified and acid pectins were distributed unevenly, with the former mainly at the tip and the latter on the distal region. Cellulose was distributed uniformly on the surface with less at the tip; callose reduced gradually from base to sub-tip of the pollen tubes and no callose at the tip of the tube was detected. B toxicity led to the deposition of esterified and acid pectins, cellulose, and callose at the tip of the pollen tube. Results from scanning electron microscopy and transmission electron microscopy showed that B toxicity also altered pollen tube wall ultrastructure. The results from enzymatic treatment illustrated that there existed a close relationship among pectins, cellulose, and callose. B toxicity also altered the relationship. In a word, B toxicity altered deposition and relationship of pectins, cellulose, and callose of pollen tube wall.

Morphological observation and protein expression of fertile and abortive ovules in Castanea mollissima.

Chinese chestnuts (Castanea mollissima Blume.) contain 12-18 ovules in one ovary, but only one ovule develops into a seed, indicating a high ovule abortion rate. In this study, the Chinese chestnut 'Huaihuang' was used to explore the possible mechanisms of ovule abortion with respect to morphology and proteomics. The morphology and microstructure of abortive ovules were found to be considerably different from those of fertile ovules at 20 days after anthesis (20 DAA). The fertile ovules had completely formed tissues, such as the embryo sac, embryo and endosperm. By contrast, in the abortive ovules, there were no embryo sacs, and wide spaces between the integuments were observed, with few nucelli. Fluorescence labelling of the nuclei and transmission electron microscopy (TEM) observations showed that cells of abortive ovules were abnormally shaped and had thickened cell walls, folded cell membranes, condensed cytoplasm, ruptured nuclear membranes, degraded nucleoli and reduced mitochondria. The iTRAQ (isobaric tag for relative and absolute quantitation) results showed that in the abortive ovules, low levels of soluble protein with small molecular weights were found, and most of differently expressed proteins (DEPs) were related to protein synthesis, accumulation of active oxygen free radical, energy synthesis and so on. These DEPs might be associated with abnormal ovules formation.

Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima).

BACKGROUND: The Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima. FINDINGS: We produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ∼785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ∼13.62 million years ago. CONCLUSIONS: The high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality.

Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut (Castanea mollissima Blume) cotyledons.

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Boron Toxicity Causes Multiple Effects on Malus domestica Pollen Tube Growth.

Boron is an important micronutrient for plants. However, boron is also toxic to cells at high concentrations, although the mechanism of this toxicity is not known. This study aimed to evaluate the effect of boron toxicity on Malus domestica pollen tube growth and its possible regulatory pathway. Our results showed that a high concentration of boron inhibited pollen germination and tube growth and led to the morphological abnormality of pollen tubes. Fluorescent labeling coupled with a scanning ion-selective electrode technique detected that boron toxicity could decrease [Ca(2+)]c and induce the disappearance of the [Ca(2+)]c gradient, which are critical for pollen tube polar growth. Actin filaments were therefore altered by boron toxicity. Immuno-localization and fluorescence labeling, together with fourier-transform infrared analysis, suggested that boron toxicity influenced the accumulation and distribution of callose, de-esterified pectins, esterified pectins, and arabinogalactan proteins in pollen tubes. All of the above results provide new insights into the regulatory role of boron in pollen tube development. In summary, boron likely plays a structural and regulatory role in relation to [Ca(2+)]c, actin cytoskeleton and cell wall components and thus regulates Malus domestica pollen germination and tube polar growth.