Genome-Wide Identification and Expression Analysis of Chitinase-like Genes in Petunia axillaris.

Chitinase (EC 3.2.1.14) is a kind of chitin-degrading glycosidase, which plays important roles in the abiotic and biotic defense of plants. In this study, we conducted whole-genome annotation, molecular evolution, and gene expression analyses on the chitinase-like (CTL) gene family members of Petunia axillaris. Thirty-three Petunia axillarischitinase-like genes (PaCTLs) were identified from the latest Petunia genome database. According to the phylogenetic analyses, these genes were divided into GH18 and GH19 subgroups and further subdivided into five classes (Class I to Class V). Conserved motif arrangements indicated their functional relevance within each group. The expansion and homeology analyses showed that gene replication events played an important role in the evolution of PaCTLs and the increase of the GH18 subgroup members was the main reason for the expansion of the PaCTL gene family in the evolution progress. By qRT-PCR analysis, we found that most of the PaCTLs showed a very low expression level in the normal growing plants. But lots of PaCTLs showed upregulated expression profiles when the plants suffered different abiotic stress conditions. Among them, five PaCTLs responded to high temperature and exhibited significantly upregulate expression level. Correspondingly, many hormone responses, as well as biotic and abiotic stress elements were found in the promoters of PaCTLs by using cis-acting element analysis. These results provide a foundation for the exploration of PaCTLs' function and enrich the evolutionary process of the CTL gene family.

Role of gibberellin and its three GID1 receptors in Jasminum sambac stem elongation and flowering.

MAIN CONCLUSION: Taken together, our results establish a reciprocal relationship between vine elongation and flowering, and reveal that GA is a positive signal for stem elogation but a negative regulator of flowering in this species. Vines or climbing plants exhibit vigorous vegetative shoot extension. GA have long been recognized as an important signal for seasonal stem elongation and flowering in many woody perennials. However, less is explored as how GA pathway is involved in the regulation of shoot extension in woody vines. Here, we investigated the role of GA and its signaling components in shoot elongation in Jasminum sambac. We found high accumulation of GA4 in the elongating internode, in contrast to a depletion of GAs in the floral differentiating shoot, which in turn featured a higher zeatin content, and a lower IAA and JA concentrations. This GA accumulation was coincident with the strong expression of JsGA20ox1 and JsGAS1 in the leaves, as well as of the JsGA2ox3 in the internode. Treatment of GA biosynthesis inhibitor reduced elongation while stimulated the terminal flowering. Remarkably, three B-type GA-receptor genes were abundantly expressed in both internodes and leaves of the extending shoots, which could enhance GA responsiveness in heterologous transgenic Arabidopsis. Furthermore, these JsGID1s showed distinct GA-dependent interaction with the JsDELLA in a yeast-two-hybrid assay. Taken together, our results establish a reciprocal relationship between vine elongation and flowering, and reveal that GA is a positive signal for stem elogation but a negative regulator of flowering in this species.

Characterization of Two BAHD Acetyltransferases Highly Expressed in the Flowers of Jasminum sambac (L.) Aiton.

Volatile benzenoid compounds are found in diverse aromatic bouquets emitted by most moth-pollinated flowers. The night-blooming Jasminum sambac is widely cultivated worldwide in the tropics and subtropics for ornamental and industrial purposes owing to its fragrant flowers. Benzylacetate is a characteristic constituent in jasmine scent which makes up to approximately 20-30% of the total emission in the headspace or extract, but the biosynthesis enzymes and the encoding genes have not yet been described. Here, we identify two cytosolic BAHD acyltransferases specifically expressed in the petals with a positive correlation closely to the emission pattern of the volatile benzenoids. Both JsBEAT1 and JsBEAT2 could use benzylalcohol and acetate-CoA as substrates to make benzylacetate in vitro. The recombinant GST-JsBEAT1 has an estimated apparent Km of 447.3 µM for benzylalcohol and 546.0 µM for acetate-CoA, whereas in the instance of the His-JsBEAT2, the Km values are marginally lower, being 278.7 and 317.3 µM, respectively. However, the catalytic reactions by the GST-JsBEAT1 are more efficient than that by the His-JsBEAT2, based on the steady-state kcat parameters. Furthermore, ectopic expression of JsBEAT1 and JsBEAT2 in the transgenic P. hybrida plants, driven by a flower-specific promotor, significantly enhances the biosynthesis of benzylbenzoate and benzylacetate, as well as the total VOCs.

Early transcriptional response of terpenoid metabolism to Colletotrichum gloeosporioides in a resistant wild strawberry Fragaria nilgerrensis.

Modern strawberry production is often threatened by microbe pathogens. Anthracnose is among the most prominent fungal disease caused mainly by Colletotrichum gloeosporioides and leads to large-scale losses both in quality and yield. Little is known regarding the mechanisms underlying the genetics in the strawberry-C. gloeosporioides interaction. In the current research, a wild accession 'Fragaria nilgerrensis' is used as a resistant model to study the roles of terpenoid and terpene genes in leaf response to C. gloeosporioides. We found that several terpenoids and terpene genes were up-regulated at early time points after challenged with C. gloeosporioides. Among the metabolites detected, sesquiterpenes were the most significantly accumulated compounds, increasing up to ~12-fold at 18 h post infection (hpi), followed by monoterpenes which showed a slight increase upon infection. Consistently, the time-resolved transcriptome data revealed that genes pertaining to terpenoid metabolism were rapidly up-regulated and co-expressed with signaling pathway genes relevant to defense response. Notably, quantitative real-time PCR confirmed that the expression of five terpene synthase genes (TPS) were greatly enhanced, by a factor of one to three orders of magnitude at 3-6 hpi. Our results reveal a possible link between rapidly induced terpenoid metabolism and the autoimmunity underlying anthracnose resistance in a wild strawberry species.

Sugar and Hormone Dynamics and the Expression Profiles of SUT/SUC and SWEET Sweet Sugar Transporters during Flower Development in Petunia axillaris.

Flowering is the first committed step of plant sexual reproduction. While the developing flower is a strong sink requiring large quantity of sugars from photosynthetic source tissues, this process is under-temper-spatially controlled via hormone signaling pathway and nutrient availability. Sugar transporters SUT/SUC and SWEET mediate sugars movement across membranes and play a significant role in various physiological processes, including reproductive organ development. In Petunia axillaris, a model ornamental plant, 5 SUT/SUC and 36 SWEET genes are identified in the current version of the genome. Analysis of their gene structure and chromosomal locations reveal that SWEET family is moderately expanded. Most of the transporter genes are abundantly expressed in the flower than in other organs. During the five flower developmental stages, transcript levels of PaSUT1, PaSUT3, PaSWEET13c, PaSWEET9a, PaSWEET1d, PaSWEET5a and PaSWEET14a increase with the maturation of the flower and reach their maximum in the fully open flowers. PaSWEET9c, the nectar-specific PhNEC1 orthologous, is expressed in matured and fully opened flowers. Moreover, determination of sugar concentrations and phytohormone dynamics in flowers at the five developmental stages shows that glucose is the predominant form of sugar in young flowers at the early stage but depletes at the later stage, whereas sucrose accumulates only in maturated flowers prior to the corolla opening. On the other hand, GA3 content and to a less extent IAA and zeatin decreases with the flower development; however, JA, SA and ABA display a remarkable peak at mid- or later flower developmental stage.

Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Cloning and Functional Assessments of Floral-Expressed SWEET Transporter Genes from Jasminum sambac.

Sugar transporters of the SWEET family mediate cross membrane movement of mono- and disaccharides and play vital roles in diverse physiological and pathophysiological processes, including sink-source relationship, pathogen responses, reproductive growth, and development. However, it remains to be determined how these transporters function in non-module plants of agricultural significance, given the evolutionarily diverse traits. In this study, we combined transcriptome analysis, rapid amplification of cDNA ends-cloning (RACE-cloning), expression profiling, and heterologous functional assay to identify SWEET genes that may have potential roles during flower opening and sexual reproduction in Jasminum sambac . During the anthesis, the floral organs of J. sambac express seven SWEET homologous genes from all four clades of the family. JsSWEET9 and 2 are significantly upregulated when flowers are fully opened, up to 6- and 3-fold compared to unopened buds, respectively. The other transporters, JsSWEET1, 5, 10, and 17 are also accumulated slightly at stage associated with fragrance release, whereas only the vacuole transporter JsSWEET16 showed small decrease in transcript level after anthesis. The JsSWEET5, a clade II member, is capable to complement yeast cell uptake on most tested sugar substrates with a preference for hexoses, while the clade I transporter JsSWEET1 mediates merely galactose import when expressed in yeast. Our results provide first evidence for further investigation on sugar transport and allocation during flowering and reproductive processes in J. sambac.

Expression Pattern of FT/TFL1 and miR156-Targeted SPL Genes Associated with Developmental Stages in Dendrobium catenatum.

Time to flower, a process either referring to juvenile-adult phase change or vegetative-reproductive transition, is strictly controlled by an intricate regulatory network involving at least both FT/TFL1 and the micro RNA (miR)156-regulated SPL family members. Despite substantial progresses recently achieved in Arabidopsis and other plant species, information regarding the involvement of these genes during orchid development and flowering competence is still limited. Dendrobium catenatum, a popular orchid species, exhibits a juvenile phase of at least three years. Here, through whole-genome mining and whole-family expression profiling, we analyzed the homologous genes of FT/TFL1, miR156, and SPL with special reference to the developmental stages. The FT/TFL1 family contains nine members; among them, DcHd3b transcribes abundantly in young and juvenile tissues but not in adult, contrasting with the low levels of others. We also found that mature miR156, encoded by a single locus, accumulated in large quantity in protocorms and declined by seedling development, coincident with an increase in transcripts of three of its targeted SPL members, namely DcSPL14, DcSPL7, and DcSPL18. Moreover, among the seven predicted miR156-targeted SPLs, only DcSPL3 was significantly expressed in adult plants and was associated with plant maturation. Our results might suggest that the juvenile phase change or maturation in this orchid plant likely involves both the repressive action of a TFL1-like pathway and the promotive effect from an SPL3-mediated mechanism.

Characterization of JsWOX1 and JsWOX4 during Callus and Root Induction in the Shrub Species Jasminum sambac.

Plant regeneration in vitro and the underlying molecular regulatory network are of great interest to developmental biology, and have potential applications in agriculture and biotechnology. Cell growth and re-differentiation during de novo organogenesis require the activation and reprogramming of stem cells within the stem cell niche of the tissues. The WUSCHEL-related homeobox (WOX) factors play important roles in the maintenance and regulation of plant stem cells and are involved in many developmental processes. However, in woody species such as the Jasminum sambac, little is known about the involvement of WOX genes in de novo organogenesis. Here we show that two WOXs, JsWOX4 and JsWOX1, are implicated in callus proliferation and root regeneration, respectively. The expression of both, together with another member JsWOX13, are upregulated during later stage of callus formation. The JsWOX4 is associated with callus proliferation, or cell division during the redifferentiation. The overexpression of this gene results in up-regulation of JsWOX13 and another homeobox gene. The JsWOX1 plays a role in root primordium initiation, as its overexpression leads to more rooty calli and more roots per callus. JsWOX1 also possibly acts upstream of JsWOX4 and JsWOX13 transcriptionally. Our results provide further evidence regarding the functions of WOX genes in organogenesis in a woody plant.

A comparative analysis of the evolution, expression, and cis-regulatory element of polygalacturonase genes in grasses and dicots.

Cell walls are a distinguishing characteristic of plants essential to their survival. The pectin content of primary cell walls in grasses and dicots is distinctly different. Polygalacturonases (PGs) can degrade pectins and participate in multiple developmental processes of plants. This study comprehensively compared the evolution, expression, and cis-regulatory element of PGs in grasses and dicots. A total of 577 PGs identified from five grasses and five dicots fell into seven clades. Evolutionary analysis demonstrated the distinct differences between grasses and dicots in patterns of gene duplication and loss, and evolutionary rates. Grasses generally contained much fewer clade C and F members than dicots. We found that this disparity was the result of less duplication and more gene losses in grasses. More duplications occurred in clades D and E, and expression analysis showed that most of clade E members were expressed ubiquitously at a high overall level and clade D members were closely related to male reproduction in both grasses and dicots, suggesting their biological functions were highly conserved across species. In addition to the general role in reproductive development, PGs of clades C and F specifically played roles in root development in dicots, shedding light on organ differentiation between the two groups of plants. A regulatory element analysis of clade C and F members implied that possible functions of PGs in specific biological responses contributed to their expansion and preservation. This work can improve the knowledge of PGs in plants generally and in grasses specifically and is beneficial to functional studies.

Dissecting the complex molecular evolution and expression of polygalacturonase gene family in Brassica rapa ssp. chinensis.

Polygalacturonases (PGs) participate in pectin disassembly of cell wall and belong to one of the largest hydrolase families in plants. In this study, we identified 99 PG genes in Brassica rapa. Comprehensive analysis of phylogeny, gene structures, physico-chemical properties and coding sequence evolution demonstrated that plant PGs should be classified into seven divergent clades and each clade's members had specific sequence and structure characteristics, and/or were under specific selection pressures. Genomic distribution and retention rate analysis implied duplication events and biased retention contributed to PG family's expansion. Promoter divergence analysis using "shared motif method" revealed a significant correlation between regulatory and coding sequence evolution of PGs, and proved Clades A and E were of ancient origin. Quantitative real-time PCR analysis showed that expression patterns of PGs displayed group specificities in B. rapa. Particularly, nearly half of PG family members, especially those of Clades C, D and F, closely relates to reproductive development. Most duplicates showed similar expression profiles, suggesting dosage constraints accounted for preservation after duplication. Promoter-GUS assay further indicated PGs' extensive roles and possible redundancy during reproductive development. This work can provide a scientific classification of plant PGs, dissect the internal relationships between their evolution and expressions, and promote functional researches.

BcMF26a and BcMF26b Are Duplicated Polygalacturonase Genes with Divergent Expression Patterns and Functions in Pollen Development and Pollen Tube Formation in Brassica campestris.

Polygalacturonase (PG) is one of the cell wall hydrolytic enzymes involving in pectin degradation. A comparison of two highly conserved duplicated PG genes, namely, Brassica campestris Male Fertility 26a (BcMF26a) and BcMF26b, revealed the different features of their expression patterns and functions. We found that these two genes were orthologous genes of At4g33440, and they originated from a chromosomal segmental duplication. Although structurally similar, their regulatory and intron sequences largely diverged. QRT-PCR analysis showed that the expression level of BcMF26b was higher than that of BcMF26a in almost all the tested organs and tissues in Brassica campestris. Promoter activity analysis showed that, at reproductive development stages, BcMF26b promoter was active in tapetum, pollen grains, and pistils, whereas BcMF26a promoter was only active in pistils. In the subcellular localization experiment, BcMF26a and BcMF26b proteins could be localized to the cell wall. When the two genes were co-inhibited, pollen intine was formed abnormally and pollen tubes could not grow or stretch. Moreover, the knockout mutants of At4g33440 delayed the growth of pollen tubes. Therefore, BcMF26a/b can participate in the construction of pollen wall by modulating intine information and BcMF26b may play a major role in co-inhibiting transformed plants.

Identification and expression analysis of BoMF25, a novel polygalacturonase gene involved in pollen development of Brassica oleracea.

KEY MESSAGE: BoMF25 acts on pollen wall. Polygalacturonase (PG) is a pectin-digesting enzyme involved in numerous plant developmental processes and is described to be of critical importance for pollen wall development. In the present study, a PG gene, BoMF25, was isolated from Brassica oleracea. BoMF25 is the homologous gene of At4g35670, a PG gene in Arabidopsis thaliana with a high expression level at the tricellular pollen stage. Collinear analysis revealed that the orthologous gene of BoMF25 in Brassica campestris (syn. B. rapa) genome was probably lost because of genome deletion and reshuffling. Sequence analysis indicated that BoMF25 contained four classical conserved domains (I, II, III, and IV) of PG protein. Homology and phylogenetic analyses showed that BoMF25 was clustered in Clade F. The putative promoter sequence, containing classical cis-acting elements and pollen-specific motifs, could drive green fluorescence protein expression in onion epidermal cells. Quantitative RT-PCR analysis suggested that BoMF25 was mainly expressed in the anther at the late stage of pollen development. In situ hybridization analysis also indicated that the strong and specific expression signal of BoMF25 existed in pollen grains at the mature pollen stage. Subcellular localization showed that the fluorescence signal was observed in the cell wall of onion epidermal cells, which suggested that BoMF25 may be a secreted protein localized in the pollen wall.