Variation for Resistance to <i>Alternaria tenuissima</i> and Potential Structural Mechanism among Different Cultivars of <i>Chrysanthemum morifolium</i>

Black spot disease, caused by the necrotrophic fungus Alternaria tenuissima (Fr.) Wiltsh (A. tenuissima), is considered a highly destructive disease of Chrysanthemum (Chrysanthemum morifolium Ramat.). A set of 17 accessions of commercial chrysanthemum cultivars were evaluated for resistance to A. tenuissima by seedling artificial inoculation. It was found that the reaction of the accessions to artificial inoculation ranged from resistant to highly susceptible. Five varieties of chrysanthemum (‘Zhongshan Taogui’, ‘Jinba’, ‘Zhongshan Jinguan’, ‘Jinling Wanhuang’ and ‘Jinling Yangguang’) were resistant; two varieties of chrysanthemum (‘Zhongshan Xinggui’ and ‘Zhongshan Jinkui’) were moderately resistant; and others were susceptible to various degrees, four varieties of chrysanthemum (‘Zhongshan Zihe’, ‘Zhongshan Jiuhong’, ‘Zaoyihong’ and ‘Jinling Jiaohuang’) were highly susceptible, especially. Some leaf morphological features of two resistant and two highly susceptible cultivars were further researched. Trichome density, length, height, gland size and stomata density were found to be associated with plant passive resistance. Resistant varieties that were identified in present study will be promising germplasm for exploitation of breeding programmes aimed at developing A. tenuissima-resistant cultivars and increasing genetic diversity.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.     

<i>OsMAPK6</i> Affects Male Fertility by Reducing Microspore Number and Delaying Tapetum Degradation in <i>Oryza sativa</i> L.

The mitogen-activated protein kinase (MAPK) cascade is important in stress signal transduction and plant development. In the present study, we identified a rice (Oryza sativa L.) mutant with reduced fertility, Oryza sativa mitogen-activated protein kinase 6 (osmapk6), which harbored a mutated MAPK gene. Scanning and transmission electron microscopy, quantitative RT-PCR analysis, TUNEL assays, RNA in situ hybridization, longitudinal and transverse histological sectioning, and map-based cloning were performed to characterize the osmapk6 mutant. The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells, tapetal cells, and microspores in the anther sac. Compared with the wild type, the total number of microspores was reduced in the osmapk6 mutant. The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development, which was the primary reason for the decrease in the total number of microspores. Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores. These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.     

Differential Responses of <i>NHX1</i> and <i>SOS1</i> Gene Expressions to Salinity in two <i>Miscanthus sinensis</i> Anderss. Accessions with Different Salt Tolerance

The lignocellulosic crop Miscanthus spp. has been identified as a good candidate for biomass production. The responses of Miscanthus sinensis Anderss. to salinity were studied to satisfy the needs for high yields in marginal areas and to avoid competition with food production. The results indicated that the relative advantages of the tolerant accession over the sensitive one under saline conditions were associated with restricted Na⁺ accumulation in shoots. Seedlings of two accessions (salt-tolerant ‘JM0119’ and salt-sensitive ‘JM0099’) were subjected to 0 (control), 100, 200, and 300 mM NaCl stress to better understand the salt-induced biochemical responses of genes involved in Na⁺ accumulation in M. sinensis. The adaptation responses of genes encoding for Na⁺ /H⁺ antiporters, NHX1 and SOS1 to NaCl stress were examined in JM0119 and JM0099.The cDNA sequences of genes examined were highly conserved among the relatives of M. sinensis based on the sequencing on approximate 600 bp-long cDNA fragments obtained from degenerate PCR. These salt-induced variations of gene expression investigated by quantitative real-time PCR provided evidences for insights of the molecular mechanisms of salt tolerance in M. sinensis. The expression of NHX1 was up-regulated by salt stress in JM0119 shoot and root tissues. However, it was hardly affected in JM0099 shoot tissue except for a significant increase at the 100 mM salt treatment, and it was salt-suppressed in the JM0099 root tissue. In the root tissue, the expression of SOS1 was induced by the high salt treatment in JM0119 but repressed by all salt treatments in JM0099. Thus, the remarkably higher expression of NHX1 and SOS1 were associated with the resistance to Na⁺ toxicity by regulation of the Na⁺ influx, efflux, and sequestration under different salt conditions.     

Soil Moisture Rather than Soil Nutrient Regulates the Belowground Bud Bank of Rhizomatous Species <i>Psammochloa villosa</i> in Arid Sand Dunes

In arid and semi-arid sand dune ecosystems, belowground bud bank plays an important role in population regeneration and vegetation restoration. However, the responses of belowground bud bank size and composition tosand burial and its induced changes in soil environmental factors have been rarely studied. In arid sand dunesof Northwestern China, we investigated belowground bud bank size and composition of the typical rhizomatouspsammophyte Psammochloa villosa as well as three key soil environmental factors (soil moisture, total carbon andtotal nitrogen) under different depths of sand burial. Total buds and rhizome buds increased significantly withincreasing burial depth, whereas tiller buds first increased and then decreased, with a peak value at the depthof 20–30 cm. Soil moisture increased significantly with sand burial depth, and was positively correlated withthe number of all buds and rhizome buds. Soil total carbon concentration first increased and then decreased withsand burial depth, and total nitrogen concentration was significantly lower under deep sand burial than those atshallow depths, and only the number of tiller buds was positively correlated with soil total nitrogen concentration.These results indicate that soil moisture rather than soil nutrient might regulate the belowground bud bank of P.villosa, and that clonal psammophytes could regulate their belowground bud bank in response to sand burial andthe most important environmental stress (i.e., soil moisture). These responses, as the key adaptive strategy, mayensure clonal plant population regeneration and vegetation restoration in arid sand dunes.     

Characterization and Candidate Gene Analysis of the Yellow-Green Leaf Mutant <i>ygl16</i> in Rice (<i>Oryza sativa</i> L.)

Leaf color mutants are ideal materials for studying many plant physiological and metabolic processes such as photosynthesis, photomorphogenesis, hormone physiology and disease resistance. In this study, the genetically stable yellow-green leaf mutant ygl16 was identified from mutated “Xinong 1B”. Compared with the wild type, the pigment concentration and photosynthetic capacity of the ygl16 decreased significantly. The ultrastructural observation showed that the distribution of thylakoid lamellae was irregular in ygl16 chloroplasts, and the grana and matrix lamellae were blurred and loose in varied degrees, and the chloroplast structure was disordered, while the osmiophilic corpuscles increased. The results of the genetic analysis and mapping showed that the phenotype of ygl16 was controlled by a pair of recessive nuclear gene. The gene located in the 56Kb interval between RM25654 and R3 on the long arm of chromosome 10. The sequencing results showed that the 121st base of the first intron of the candidate gene OsPORB/FGL changed from A to T in the interval. qRT-PCR results showed that the expression of chlorophyll synthase-related genes in the mutant decreased.     

Cloning and Characterization of <i>EuGID1</i> in <i>Eucommia ulmoides</i> Oliver

Gibberellic acid controlled the key developmental processes of the life cycle of landing plants, and regulated the growth and development of plants. In this study, a novel gibberellin receptor gene EuGID1 was obtained from Eucommia ulmoides Oliver. The cDNA of EuGID1 was 1556 bp, and the open reading frame was 1029 bp, which encoded 343 amino acids. EuGID1 had the homology sequence with the hormone-sensitive lipase family. Amino acid sequence alignment confirmed EuGID1 protein had the highest homology with the GID1 protein of Manihot esculenta. EuGID1 was located in the nucleus and cell membrane and had expression in four plant organs. Overexpression of EuGID1 in transgenic Arabidopsis plants promoted plant elongation and increased siliques yield.     

Identification,Isolation and Characterization of <i>GaCyPI</i> Gene in <i>Gossypium arboreum</i> under Cotton Leaf Curl Virus Disease Stress

Pakistan is facing the threat of Cotton Leaf Curl Virus (CLCuV) which is transmitted through whitefly to cotton crop. Molecular mechanism of leaf epicuticular wax protects the plants from different pathogens including insect attack and disease transmission. Objective of current study is the isolation and characterization of a wax related gene GaCyPI from Gossypium arboreum under CLCuV infection. A fragment of 475 bp was isolated from the total RNA and 3’ and 5’ RACE-PCR products were arranged by overlapping the extended sequences at both the ends. Deduced protein sequence of GaCyPI showed homology with Cyclophilin cis-trans isomerase gene of Gossypium ramondii and Gossypium barbadanse. Multiple sequence alignment also revealed homology among the coding sequences of same gene. GaCyPI protein comprised of 173 amino acids and ORF finder revealed the 69 bases upstream at 5’ while 350 bp at 3’UTR. InterProScan revealed that it belongs to Cyclophilin-type peptidyl prolyl cis/trans isomerase (PPIase) family. Active sites are visible at specific amino acid positions and 3D structure was stable in Ramachandran plot. Prosa server showed protein residues have average 3D-1D score >= 0.2 and Z-Score was −6.74. Phylogenetic analysis revealed that G. raimondii is the closest species that shares the same sequence. Hence, GaCyPI has strong role in plants’ epicuticular wax and its genetic transformation may protect the cotton from whitefly which transmits CLCuV.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Genome-Wide Analysis of the <i>KANADI</i> Gene Family and Its Expression Patterns under Different Nitrogen Concentrations Treatments in <i>Populus trichocarpa</i>

KANADI (KAN) is a plant-specific gene that controlled the polarity development of lateral organs. It mainly acted on the abaxial characteristics of plants to make the lateral organs asymmetrical. However, it had been less identified in woody plants. In this study, the members of the KAN gene family in Populus trichocarpa were identified and analyzed using the bioinformatics method. The results showed that a total of 8 KAN family members were screened out, and each member contained the unique GARP domain and conserved region of the family proteins. Phylogenetic analysis and their gene structures revealed that all KAN genes from P. trichocarpa, Arabidopsis thaliana, and Nicotiana benthamiana could be divided into four subgroups, while the eight genes in P. trichocarpa were classified into three subgroups, respectively. The analysis of tissue-specific expression indicated that PtKAN1 was highly expressed in young leaves, PtKAN6 was highly expressed in young leaves and mature leaves, PtKAN2, PtKAN5, and PtKAN7 were highly expressed in nodes and internodes, PtKAN8 was highly expressed in roots, and PtKAN3 and PtKAN4 showed low expression levels in all tissues. Among them, PtKAN2 and PtKAN6, and PtKAN4 and PtKAN5 might have functional redundancy. Under high nitrogen concentrations, PtKAN2 and PtKAN8 were highly expressed in mature stems and leaves, respectively, while PtKAN4, PtKAN5, and PtKAN7 were highly expressed in roots. This study laid a theoretical foundation for further study of the KAN gene-mediated nitrogen effect on root development.     

Phytosulfokine-α Promotes Root Growth by Repressing Expression of <i>Pectin Methylesterase Inhibitor</i> (<i>PMEI</i>) Genes in <i>Medicago truncatula</i>

Phytosulfokine-α (PSK-α), a sulfated pentapeptide with the sequence YIYTQ, is encoded by a small precursor gene family in Arabidopsis. PSK-α regulates multiple growth and developmental processes as a novel peptide hormone. Despite its importance, functions of PSK-α in M. truncatula growth remains unknown. In this study, we identified five genes to encode PSK-α precursors in M. truncatula. All of these precursors possess conserved PSK-α signature motif. Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root. Treatment of M. truncatula seedlings with synthetic PSK- α peptide significantly promoted root elongation. In addition, expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-α signaling might regulate cell wall structure via PMEI-PME module to promote root cell growth. Taken together, our results shed light on the mechanism by which PSK-α promotes root growth in M. truncatula, providing a new resource for improvement of root growth in agriculture.