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1.  The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90) 
Journal of Experimental Botany  2014;65(8):2147-2160.
Summary
NtCOI1, a tobacco orthologue of the Arabidopsis jasmonate co-receptor COI1, was shown to regulate primary carbohydrate metabolism in both reproductive and vegetative organs of Nicotiana tabacum L. cv. TN90.
Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.
doi:10.1093/jxb/eru084
PMCID: PMC3991746  PMID: 24604735
COI1; jasmonate; primary metabolism; starch; tobacco.
2.  A Regulatory Network for Coordinated Flower Maturation 
PLoS Genetics  2012;8(2):e1002506.
For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.
Author Summary
Perfect flowers have both male organs that produce and release pollen and female organs that make and harbor seeds. Flowers also often attract pollinators using visual or chemical signals. So that male, female, and pollinator attraction functions occur at the right time, flower organs must grow and mature in a coordinated fashion. In the model self-pollinating plant Arabidopsis, a transcriptional network regulates genes that ensure coordinated growth of different flower organs, as well as pollen release and gynoecium (female) competence to support pollination. This network also regulates nectary development and production of volatile chemicals that may attract or repel insects. We have studied growth, chemical signal levels, and gene expression in mutants affected in components of this network, in order to determine how flower growth is controlled. Several plant hormones act in a cascade that promotes flower maturation. Moreover, regulatory feedback loops affect the timing and extent of developmental steps. Positive feedbacks may ensure that the development of different flower organs is coordinated and rapid, whereas negative feedbacks may allow growth to cease once flowers have opened. Our results provide a framework to understand how flower opening and reproduction are coordinated in Arabidopsis and other flowering plants.
doi:10.1371/journal.pgen.1002506
PMCID: PMC3276552  PMID: 22346763
3.  A flavonoid 3-O-glucoside:2″-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana 
The Plant Journal  2014;79(5):769-782.
Flavonol 3-O-diglucosides with a 1→2 inter-glycosidic linkage are representative pollen-specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild-type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild-type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP-glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen-specific flavonol structure. Kaempferol and quercetin 3-O-glucosyl-(1→2)-glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild-type plants. Recombinant UGT79B6 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucosyl-(1→2)-glucoside. UGT79B6 recognized 3-O-glucosylated/galactosylated anthocyanins/flavonols but not 3,5- or 3,7-diglycosylated flavonoids, and prefers UDP-glucose, indicating that UGT79B6 encodes flavonoid 3-O-glucoside:2″-O-glucosyltransferase. A UGT79B6-GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.
doi:10.1111/tpj.12580
PMCID: PMC4282749  PMID: 24916675
glucosyltransferase; At5g54010; NM_124780; tapetum; pollen; glycosyltransferase; flavonol; flavonoid; Arabidopsis thaliana
4.  Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana 
Plant Signaling & Behavior  2011;6(1):66-71.
Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.
doi:10.4161/psb.6.1.13999
PMCID: PMC3122008  PMID: 21301212
cytokinin; cytokinin receptor; female gametophyte; male gametophyte; stigma
5.  Identifying the transporters of different flavonoids in plants 
Plant Signaling & Behavior  2010;5(7):860-863.
We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT's substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologs within the large Arabidopsis MATE family.
doi:10.4161/psb.5.7.11894
PMCID: PMC3115035  PMID: 20505354
auxin; flavonoid; guard cell; pollen; transporter
6.  Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots 
The New Phytologist  2013;201(2):466-475.
Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT.Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [3H]-indole-3-acetic acid tracer.The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport.These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.
doi:10.1111/nph.12558
PMCID: PMC4260840  PMID: 24251900
Arabidopsis thaliana; flavonol biosynthesis; flavonol glycoside; flavonol glycosyltransferases; plant growth; polar auxin transport
7.  Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway 
BMC Research Notes  2010;3:255.
Background
The coordinated activity of different flavonoid biosynthesis genes in Arabidopsis thaliana results in tissue-specific accumulation of flavonols, anthocyanins and proanthocyanidins (PAs). These compounds possess diverse functions in plants including light-attenuation and oxidative stress protection. Flavonoids accumulate in a stimulus- and/or development-dependent manner in specific parts of the plant. PAs accumulate in the seed coat (testa).
Findings
We describe the biological material and the preparation of total RNA for the AtGenExpress developmental silique and seed series. AtGenExpress ATH1 GeneChip expression data from the different stages were reanalyzed and verified using quantitative real time PCR (qPCR). We observed organ-specific transcript accumulation of specific flavonoid biosynthetic genes consistent with previously published data and our PA compound accumulation data. In addition, we investigated the regulation of PA accumulation in developing A. thaliana seeds by correlating gene expression patterns of specific flavonoid biosynthesis genes with different seed embryonic developmental stages and organs and present two useful marker genes for isolated valve and replum organs, as well as one seed-specific marker.
Conclusions
Potential caveats of array-based expression data are discussed based on comparisons with qPCR data. Results from ATH1 microarray and qPCR experiments revealed a shift in gene activity from general flavonoid biosynthesis at early stages of seed development to PA synthesis at late (mature) stages of embryogenesis. The examined PA accumulation-associated genes, including biosynthetic and regulatory genes, were found to be exclusively expressed in immature seeds. Accumulation of PAs initiates at the early heart stage of silique and seed development. Our findings provide new insights for further studies targeting the PA pathway in seeds.
doi:10.1186/1756-0500-3-255
PMCID: PMC2958888  PMID: 20929528
8.  An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development 
BMC Plant Biology  2013;13:176.
Background
Red coloration of fruit is an important trait in apple, and it is mainly attributed to the accumulation of anthocyanins, a class of plant flavonoid metabolites. Anthocyanin biosynthesis is genetically determined by structural and regulatory genes. Plant tissue pigmentation patterns are mainly controlled by expression profiles of regulatory genes. Among these regulatory genes are MYB transcription factors (TFs), wherein the class of two-repeats (R2R3) is deemed the largest, and these are associated with the anthocyanin biosynthesis pathway. Although three MdMYB genes, almost identical in nucleotide sequences, have been identified in apple, it is likely that there are other R2R3 MYB TFs that are present in the apple genome that are also involved in the regulation of coloration of red color pigmentation of the skin of apple fruits.
Results
In this study, a novel R2R3 MYB gene has been isolated and characterized in apple. This MYB gene is closely related to the Arabidopsis thaliana AtMYB3, and has been designated as MdMYB3. This TF belongs to the subgroup 4 R2R3 family of plant MYB transcription factors. This apple MdMYB3 gene is mapped onto linkage group 15 of the integrated apple genetic map. Transcripts of MdMYB3 are detected in all analyzed tissues including leaves, flowers, and fruits. However, transcripts of MdMYB3 are higher in excocarp of red-skinned apple cultivars than that in yellowish-green skinned apple cultivars. When this gene is ectopically expressed in Nicotiana tabacum cv. Petite Havana SR1, flowers of transgenic tobacco lines carrying MdMYB3 have exhibited increased pigmentation and accumulate higher levels of anthocyanins and flavonols than wild-type flowers. Overexpression of MdMYB3 has resulted in transcriptional activation of several flavonoid pathway genes, including CHS, CHI, UFGT, and FLS. Moreover, peduncles of flowers and styles of pistils of transgenic plants overexpressing MdMYB3 are longer than those of wild-type plants, thus suggesting that this TF is involved in regulation of flower development.
Conclusions
This study has identified a novel MYB transcription factor in the apple genome. This TF, designated as MdMYB3, is involved in transcriptional activation of several flavonoid pathway genes. Moreover, this TF not only regulates the accumulation of anthocyanin in the skin of apple fruits, but it is also involved in the regulation of flower development, particularly that of pistil development.
doi:10.1186/1471-2229-13-176
PMCID: PMC3833268  PMID: 24199943
Anthocyanin; Apple; MYB transcription factors; Malus × domestica; Flavonoids; Reproductive tissues
9.  Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells 
Journal of Experimental Botany  2009;61(1):41-53.
To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5′-flanking region, including the promoter and 5′-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.
doi:10.1093/jxb/erp280
PMCID: PMC2791113  PMID: 19861654
ADF; cotton; F-actin; gene expression; pollen development
10.  The NAC-like gene ANTHER INDEHISCENCE FACTOR acts as a repressor that controls anther dehiscence by regulating genes in the jasmonate biosynthesis pathway in Arabidopsis  
Journal of Experimental Botany  2013;65(2):621-639.
ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, β-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7–9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.
doi:10.1093/jxb/ert412
PMCID: PMC3904717  PMID: 24323506
Anther dehiscence; ANTHER INDEHISCENCE FACTOR; jasmonate signalling; NAC-like gene; repressor.
11.  Factors affecting quantity of pollen dispersal of spray cut chrysanthemum (Chrysanthemum morifolium) 
BMC Plant Biology  2014;14:5.
Background
Spray cut chrysanthemum is a vital flower with high ornamental value and popularity in the world. However, the excessive quantity of pollen dispersal of most spray cut chrysanthemum is an adverse factor during its flowering stage, and can significantly reduce its ornamental value and quickly shorten its vase life. More seriously, excessive pollen grains in the air are usually harmful to people, especially for those with pollen allergies. Therefore, in order to obtain some valuable information for developing spray cut chrysanthemum with less-dispersed or non-dispersed pollen in the future breeding programs, we here investigated the factors affecting quantity of pollen dispersal of spray cut chrysanthemum with four cultivars, i.e. ‘Qx-097’, ‘Noa’, ‘Qx-115’, and ‘Kingfisher’, that have different quantity of pollen dispersal.
Results
‘Qx-097’ with high quantity of pollen dispersal has 819 pollen grains per anther, 196.4 disk florets per inflorescence and over 800,000 pollen grains per inflorescence. The corresponding data for ‘Noa’ with low quantity of pollen dispersal are 406, 175.4 and over 350,000, respectively; and 219, 144.2 and nearly 160,000 for ‘Qx-115’ without pollen dispersal, respectively. ‘Kingfisher’ without pollen dispersal has 202.8 disk florets per inflorescence, but its anther has no pollen grains. In addition, ‘Qx-097’ has a very high degree of anther cracking that nearly causes a complete dispersal of pollen grains from its anthers. ‘Noa’ has a moderate degree of anther cracking, and pollen grains in its anthers are not completely dispersed. However, the anthers of ‘Qx-115’ and ‘Kingfisher’ do not crack at all. Furthermore, microsporogenesis and pollen development are normal in ‘Qx-097’, whereas many microspores or pollen degenerate in ‘Noa’, most of them abort in ‘Qx-115’, and all of them degrade in ‘Kingfisher’.
Conclusions
These results suggest that quantity of pollen dispersal in spray cut chrysanthemum are mainly determined by pollen quantity per anther, and capacity of pollen dispersal. Abnormality during microsporogenesis and pollen development significantly affects pollen quantity per anther. Capacity of pollen dispersal is closely related to the degree of anther dehiscence. The entire degeneration of microspore or pollen, or the complete failure of anther dehiscence can cause the complete failure of pollen dispersal.
doi:10.1186/1471-2229-14-5
PMCID: PMC3890635  PMID: 24393236
12.  Prediction of components of the sporopollenin synthesis pathway in peach by genomic and expression analyses 
BMC Genomics  2013;14:40.
Background
The outer cell wall of the pollen grain (exine) is an extremely resistant structure containing sporopollenin, a mixed polymer made up of fatty acids and phenolic compounds. The synthesis of sporopollenin in the tapetal cells and its proper deposition on the pollen surface are essential for the development of viable pollen. The beginning of microsporogenesis and pollen maturation in perennial plants from temperate climates, such as peach, is conditioned by the duration of flower bud dormancy. In order to identify putative genes involved in these processes, we analyzed the results of previous genomic experiments studying the dormancy-dependent gene expression in different peach cultivars.
Results
The expression of 50 genes induced in flower buds after the endodormancy period (flower-bud late genes) was compared in ten cultivars of peach with different dormancy behaviour. We found two co-expression clusters enriched in putative orthologs of sporopollenin synthesis and deposition factors in Arabidopsis. Flower-bud late genes were transiently expressed in anthers coincidently with microsporogenesis and pollen maturation processes. We postulated the participation of some flower-bud late genes in the sporopollenin synthesis pathway and the transcriptional regulation of late anther development in peach.
Conclusions
Peach and the model plant Arabidopsis thaliana show multiple elements in common within the essential sporopollenin synthesis pathway and gene expression regulatory mechanisms affecting anther development. The transcriptomic analysis of dormancy-released flower buds proved to be an efficient procedure for the identification of anther and pollen development genes in perennial plants showing seasonal dormancy.
doi:10.1186/1471-2164-14-40
PMCID: PMC3556096  PMID: 23331975
13.  Auxin polar transport in stamen formation and development: how many actors? 
In flowering plants, proper development of stamens, the male reproductive organs, is required for successful sexual reproduction. In Arabidopsis thaliana normally six stamen primordia arise in the third whorl of floral organs and subsequently differentiate into stamen filaments and anthers, where male meiosis occurs, thus ending the early developmental phase. This early phase is followed by a late developmental phase, which consists of a rapid elongation of stamen filaments coordinated with anther dehiscence and pollen maturation, and terminates with mature pollen grain release at anthesis. Increasing evidence suggests that auxin transport is necessary for both early and late phases of stamen development. It has been shown that different members of PIN (PIN-FORMED) family are involved in the early phase, whereas members of both PIN and P-glycoproteins of the ABCB (PGP) transporter families are required during the late developmental phase. In this review we provide an overview of the increasing knowledge on auxin transporters involved in Arabidopsis stamen formation and development and we discuss their role and functional conservation across plant species.
doi:10.3389/fpls.2014.00333
PMCID: PMC4100440  PMID: 25076953
stamen development; auxin transport; Arabidopsis; dicots; monocots
14.  Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato 
Journal of Experimental Botany  2014;65(9):2507-2520.
Summary
Investigations into the role of tomato ARF6 and ARF8 reveal that they are critical components in floral and gynoecium development before anthesis.
Auxin regulates the expression of diverse genes that affect plant growth and development. This regulation requires AUXIN RESPONSE FACTORS (ARFs) that bind to the promoter regions of these genes. ARF6 and ARF8 in Arabidopsis thaliana are required to promote inflorescence stem elongation and late stages of petal, stamen, and gynoecium development. All seed plants studied thus far have ARF6 and ARF8 orthologues as well as the microRNA miR167, which targets ARF6 and ARF8. Whether these genes have broadly conserved roles in flower development is not known. To address this question, the effects of down-regulation of ARF6 and ARF8 were investigated through transgenic expression of Arabidopsis MIR167a in tomato, which diverged from Arabidopsis before the radiation of dicotyledonous plants approximately 90–112 million years ago. The transgenic tomato plants overexpressing MIR167a exhibited reductions in leaf size and internode length as well as shortened petals, stamens, and styles. More significantly, the transgenic plants were female-sterile as a result of failure of wild-type pollen to germinate on the stigma surface and/or to grow through the style. RNA-Seq analysis identified many genes with significantly altered expression patterns, including those encoding products with functions in ‘transcription regulation’, ‘cell wall’ and ‘lipid metabolism’ categories. Putative orthologues of a subset of these genes were also differentially expressed in Arabidopsis arf6 arf8 mutant flowers. These results thus suggest that ARF6 and ARF8 have conserved roles in controlling growth and development of vegetative and flower organs in dicots.
doi:10.1093/jxb/eru141
PMCID: PMC4036516  PMID: 24723401
ARF6; ARF8; expression; female sterility; flower development; tomato.
15.  Is There ‘Anther-Anther Interference’ within a Flower? Evidences from One-by-One Stamen Movement in an Insect-Pollinated Plant 
PLoS ONE  2014;9(1):e86581.
The selective pressure imposed by maximizing male fitness (pollen dispersal) in shaping floral structures is increasingly recognized and emphasized in current plant sciences. To maximize male fitness, many flowers bear a group of stamens with temporally separated anther dehiscence that prolongs presentation of pollen grains. Such an advantage, however, may come with a cost resulting from interference of pollen removal by the dehisced anthers. This interference between dehisced and dehiscing anthers has received little attention and few experimental tests to date. Here, using one-by-one stamen movement in the generalist-pollinated Parnassia palustris, we test this hypothesis by manipulation experiments in two years. Under natural conditions, the five fertile stamens in P. palustris flowers elongate their filaments individually, and anthers dehisce successively one-by-one. More importantly, the anther-dehisced stamen bends out of the floral center by filament deflexion before the next stamen's anther dehiscence. Experimental manipulations show that flowers with dehisced anther remaining at the floral center experience shorter (1/3–1/2 less) visit durations by pollen-collecting insects (mainly hoverflies and wasps) because these ‘hungry’ insects are discouraged by the scant and non-fresh pollen in the dehisced anther. Furthermore, the dehisced anther blocks the dehiscing anther's access to floral visitors, resulting in a nearly one third decrease in their contact frequency. As a result, pollen removal of the dehiscing anther decreases dramatically. These results provide the first direct experimental evidence that anther-anther interference is possible in a flower, and that the selection to reduce such interferences can be a strong force in floral evolution. We also propose that some other floral traits, usually interpreted as pollen dispensing mechanisms, may function, at least partially, as mechanisms to promote pollen dispersal by reducing interferences between dehisced and dehiscing anthers.
doi:10.1371/journal.pone.0086581
PMCID: PMC3903572  PMID: 24475150
16.  Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers 
Journal of Experimental Botany  2012;63(18):6505-6517.
Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3′-hydroxylase (F3′H), but not the late biosynthetic gene, flavonoid 3′,5′-hydroxylase (F3′5′H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.
doi:10.1093/jxb/ers306
PMCID: PMC3504500  PMID: 23125348
Early flavonoid biosynthesis; flavone; flower colour; Japanese gentian; R2R3-MYB; transcription factor
17.  Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil 
PLoS Genetics  2009;5(8):e1000621.
Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
Author Summary
For successful reproduction in flowering plants, a single-celled pollen tube must rapidly extend through female pistil tissue, locate female gametes, and deliver sperm. Pollen tubes undergo a dramatic transformation while growing in the pistil; they grow faster compared to tubes grown in vitro and become competent to perceive and respond to navigation cues secreted by the pistil. The genes expressed by pollen tubes in response to growth in the pistil have not been characterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. Importantly, we identify a set of genes that are specifically expressed in pollen tubes in response to their growth in the pistil and are not expressed during other stages of pollen or plant development. We analyzed mutants in 33 pollen tube–expressed genes using a sensitive series of pollen function assays and demonstrate that seven of these genes are critical for pollen tube growth; two specifically disrupt growth in the pistil. By identifying pollen tube genes induced by the pistil and describing a mutant analysis scheme to understand their function, we lay the foundation for functional genomic analysis of pollen–pistil interactions.
doi:10.1371/journal.pgen.1000621
PMCID: PMC2726614  PMID: 19714218
18.  Cold stress alters transcription in meiotic anthers of cold tolerant chickpea (Cicer arietinum L.) 
BMC Research Notes  2014;7(1):717.
Background
Cold stress at reproductive phase in susceptible chickpea (Cicer arietinum L.) leads to pollen sterility induced flower abortion. The tolerant genotypes, on the other hand, produce viable pollen and set seed under cold stress. Genomic information on pollen development in cold-tolerant chickpea under cold stress is currently unavailable.
Results
DDRT-PCR analysis was carried out to identify anther genes involved in cold tolerance in chickpea genotype ICC16349 (cold-tolerant). A total of 9205 EST bands were analyzed. Cold stress altered expression of 127 ESTs (90 up-regulated, 37 down-regulated) in anthers, more than two third (92) of which were novel with unknown protein identity and function. Remaining about one third (35) belonged to several functional categories such as pollen development, signal transduction, ion transport, transcription, carbohydrate metabolism, translation, energy and cell division. The categories with more number of transcripts were carbohydrate/triacylglycerol metabolism, signal transduction, pollen development and transport. All but two transcripts in these categories were up-regulated under cold stress. To identify time of regulation after stress and organ specificity, expression levels of 25 differentially regulated transcripts were also studied in anthers at six time points and in four organs (anthers, gynoecium, leaves and roots) at four time points.
Conclusions
Limited number of genes were involved in regulating cold tolerance in chickpea anthers. Moreover, the cold tolerance was manifested by up-regulation of majority of the differentially expressed transcripts. The anthers appeared to employ dual cold tolerance mechanism based on their protection from cold by enhancing triacylglycerol and carbohydrate metabolism; and maintenance of normal pollen development by regulating pollen development genes. Functional characterization of about two third of the novel genes is needed to have precise understanding of the cold tolerance mechanisms in chickpea anthers.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-717) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-0500-7-717
PMCID: PMC4201710  PMID: 25306382
Anthers; Cicer arietinum; Cold stress; Cold tolerance; Gene expression; Male gametophyte; Pollen
19.  Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana 
Journal of Experimental Botany  2009;60(3):751-763.
Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants.
doi:10.1093/jxb/ern323
PMCID: PMC2652062  PMID: 19129166
Aglycone; Arabidopsis; auxin; ethylene; flavonoid; plant architecture
20.  Up and down: stamen movements in Ruta graveolens (Rutaceae) enhance both outcrossing and delayed selfing 
Annals of Botany  2012;110(5):1017-1025.
Background and Aims
Stamen movements directly determine pollen fates and mating patterns by altering positions of female and male organs. However, the implications of such movements in terms of pollination are not well understood. Recently, complex patterns of stamen movements have been identified in Loasaceae, Parnassiaceae, Rutaceae and Tropaeolaceae. In this study the stamen movements in Ruta graveolens (Rutaceae) and their impact on pollination are determined.
Methods
Pollination effects of stamen movements were studied in Ruta graveolens, in which one-by-one uplifting and falling back is followed by simultaneous movement of all stamens in some flowers. Using 30 flowers, one stamen was manipulated either to be immobilized or to be allowed to move freely towards the centre of the flower but be prevented from falling back. Pollen loads on stigmas and ovule fertilization in flowers with or without simultaneous stamen movement were determined.
Results
Pollen removal decreased dramatically (P < 0·001) when the stamen was stopped from uplifting because its anther was seldom contacted by pollinators. When a stamen stayed at the flower's centre, pollen removal of the next freely moved anther decreased significantly (P < 0·005) because of fewer touches by pollinators and quick leaving of pollinators that were discouraged by the empty anther. Simultaneous stamen movement occurred only in flowers with low pollen load on the stigma and the remaining pollen in anthers dropped onto stigma surfaces after stamens moved to the flower's centre.
Conclusions
In R. graveolens pollen removal is promoted through one-by-one movement of the stamen, which presents pollen in doses to pollinators by successive uplifting of the stamen and avoids interference of two consecutively dehisced anthers by falling back of the former stamen before the next one moves into the flower's centre. Simultaneous stamen movement at the end of anthesis probably reflects an adaptation for late-acting self-pollination.
doi:10.1093/aob/mcs181
PMCID: PMC3448434  PMID: 22875813
Dichogamy; experimental manipulation; pollen presentation; pollen removal; pollination; reproductive assurance; Ruta graveolens; stamen movement
21.  AMS-dependent and independent regulation of anther transcriptome and comparison with those affected by other Arabidopsis anther genes 
BMC Plant Biology  2012;12:23.
Background
In flowering plants, the development of male reproductive organs is controlled precisely to achieve successful fertilization and reproduction. Despite the increasing knowledge of genes that contribute to anther development, the regulatory mechanisms controlling this process are still unclear.
Results
In this study, we analyzed the transcriptome profiles of early anthers of sterile mutants aborted microspores (ams) and found that 1,368 genes were differentially expressed in ams compared to wild type anthers, affecting metabolism, transportation, ubiquitination and stress response. Moreover, the lack of significant enrichment of potential AMS binding sites (E-box) in the promoters of differentially expressed genes suggests both direct and indirect regulation for AMS-dependent regulation of anther transcriptome involving other transcription factors. Combining ams transcriptome profiles with those of two other sterile mutants, spl/nzz and ems1/exs, expression of 3,058 genes were altered in at least one mutant. Our investigation of expression patterns of major transcription factor families, such as bHLH, MYB and MADS, suggested that some closely related homologs of known anther developmental genes might also have similar functions. Additionally, comparison of expression levels of genes in different organs suggested that anther-preferential genes could play important roles in anther development.
Conclusion
Analysis of ams anther transcriptome and its comparison with those of spl/nzz and ems1/exs anthers uncovered overlapping and distinct sets of regulated genes, including those encoding transcription factors and other proteins. These results support an expanded regulatory network for early anther development, providing a series of hypotheses for future experimentation.
doi:10.1186/1471-2229-12-23
PMCID: PMC3305669  PMID: 22336428
22.  Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling 
The Plant Journal   2007;50(4):660-677.
The genes MYB11, MYB12 and MYB111 share significant structural similarity and form subgroup 7 of the Arabidopsis thaliana R2R3-MYB gene family. To determine the regulatory potential of these three transcription factors, we used a combination of genetic, functional genomics and metabolite analysis approaches. MYB11, MYB12 and MYB111 show a high degree of functional similarity and display very similar target gene specificity for several genes of flavonoid biosynthesis, including CHALCONE SYNTHASE, CHALCONE ISOMERASE, FLAVANONE 3-HYDROXYLASE and FLAVONOL SYNTHASE1. Seedlings of the triple mutant myb11 myb12 myb111, which genetically lack a complete subgroup of R2R3-MYB genes, do not form flavonols while the accumulation of anthocyanins is not affected. In developing seedlings, MYB11, MYB12 and MYB111 act in an additive manner due to their differential spatial activity; MYB12 controls flavonol biosynthesis mainly in the root, while MYB111 controls flavonol biosynthesis primarily in cotyledons. We identified and confirmed additional target genes of the R2R3-MYB subgroup 7 factors, including the UDP-glycosyltransferases UGT91A1 and UGT84A1, and we demonstrate that the accumulation of distinct and structurally identified flavonol glycosides in seedlings correlates with the expression domains of the different R2R3-MYB factors. Therefore, we refer to these genes as PFG1–3 for ‘PRODUCTION OF FLAVONOL GLYCOSIDES’.
doi:10.1111/j.1365-313X.2007.03078.x
PMCID: PMC1976380  PMID: 17419845
flavonoid biosynthesis; R2R3-MYB; PRODUCTION OF FLAVONOL GLYCOSIDES; gene regulation; Arabidopsis
23.  Fused lobed anther and hooked stigma affect pollination, fertilization and fruit set in mango 
Plant Signaling & Behavior  2013;8(3):e23167.
Mango malformation is the most threaten disease that limits mango production, worldwide. For a long time, due to its complex nature, the cause and causal agents were strongly disputed. Diverse Fusaria, including Fusarium mangiferae, are known to be associated with the disease. There are indications that augmented level of endogenous ethylene in response to various abiotic and biotic stresses alters the morphology of reproductive organs. Here, scanning electron microscopy (SEM) of healthy and malformed reproductive organs of mango cv. Baramasi was performed to compare the functional morphology. The SEM study revealed that anthers of hermaphrodite healthy flowers were bilobed with large number of turgid pollen grains whereas malformed flowers showed fused lobed anthers with scanty deformed pollen grains. Furthermore, the stigma of healthy flowers exhibited a broad landing pad as compared to malformed stigma which showed hooked and pointed tip. All these impaired morphology of male and female reproductive organs lead to failure of sexual reproduction. This is the first evidence to show fused lobed anther with impaired pollen grains and hooked stigma with poor stigmatic receptivity are mainly responsible for restricting the pollen germination and pollen tube growth. Here we suggest that abnormal development of anthers and pistils is due to endogenously produced stress ethylene. Further, added load of cyanide, a byproduct of ethylene biosynthesis, may also contribute to the development of necrosis which lead to desiccation of anther and pistil during hypersensitive response of plants.
doi:10.4161/psb.23167
PMCID: PMC3676485  PMID: 23299320
Anther; mango malformation; pollen; scanning electron microscopy; stigma; stress ethylene
24.  Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera 
Journal of Experimental Botany  2009;60(3):853-867.
Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.
doi:10.1093/jxb/ern336
PMCID: PMC2652055  PMID: 19129169
bHLH; flavonoids; grape; leaf removal; MYB12; PAR; sugar; source; sink; WDR
25.  A R2R3-MYB Transcription Factor from Epimedium sagittatum Regulates the Flavonoid Biosynthetic Pathway 
PLoS ONE  2013;8(8):e70778.
Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.
doi:10.1371/journal.pone.0070778
PMCID: PMC3731294  PMID: 23936468

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