Evidence that an evolutionary transition from dehiscent to indehiscent fruits in Lepidium (Brassicaceae) was caused by a change in the control of valve margin identity genes

In the Brassicaceae, indehiscent fruits evolved from dehiscent fruits several times independently. Here we use closely related wild species of the genus Lepidium as a model system to analyse the underlying developmental genetic mechanisms in a candidate gene approach. ALCATRAZ (ALC), INDEHISCENT (IND), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are known fruit developmental genes of Arabidopsis thaliana that are expressed in the fruit valve margin governing dehiscence zone formation. Comparative expression analysis by quantitative RT‐PCR, Northern blot and in situ hybridization show that their orthologues from Lepidium campestre (dehiscent fruits) are similarly expressed at valve margins. In sharp contrast, expression of the respective orthologues is abolished in the corresponding tissue of indehiscent Lepidium appelianum fruits, indicating that changes in the genetic pathway identified in A. thaliana caused the transition from dehiscent to indehiscent fruits in the investigated species. As parallel mutations in different genes are quite unlikely, we conclude that the changes in gene expression patterns are probably caused by changes in upstream regulators of ALC, IND and SHP1/2, possible candidates from A. thaliana being FRUITFULL (FUL), REPLUMLESS (RPL) and APETALA2 (AP2). However, neither expression analyses nor functional tests in transgenic plants provided any evidence that the FUL or RPL orthologues of Lepidium were involved in evolution of fruit indehiscence in Lepidium. In contrast, stronger expression of AP2 in indehiscent compared to dehiscent fruits identifies AP2 as a candidate gene that deserves further investigation.     

Temporal,but not spatial,changes in expression patterns of petal identity genes are associated with loss of papillate conical cells and the shift to bird pollination in Macaronesian Lotus (Leguminosae)

• In the generally bee‐pollinated genus Lotus a group of four species have evolved bird‐pollinated flowers. The floral changes in these species include altered petal orientation, shape and texture. In Lotus these characters are associated with dorsiventral petal identity, suggesting that shifts in the expression of dorsal identity genes may be involved in the evolution of bird pollination. Of particular interest is Lotus japonicus CYCLOIDEA 2 (LjCYC2), known to determine the presence of papillate conical cells on the dorsal petal in L. japonicus. Bird‐pollinated species are unusual in not having papillate conical cells on the dorsal petal.
• Using RT‐PCR at various stages of flower development, we determined the timing of expression in all petal types for the three putative petal identity genes (CYC‐like genes) in different species with contrasting floral morphology and pollination syndromes.
• In bird‐pollinated species the dorsal identity gene, LjCYC2, is not expressed at the floral stage when papillate conical cells are normally differentiating in bee‐pollinated species. In contrast, in bee‐pollinated species, LjCYC2 is expressed during conical cell development.
• Changes in the timing of expression of the above two genes are associated with modifications in petal growth and lateralisation of the dorsal and ventral petals in the bird‐pollinated species. This study indicates that changes in the timing, rather than spatial distribution, of expression likely contribute to the modifications of petal micromorphology and petal size during the transition from bee to bird pollination in Macaronesian Lotus species.

     

A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

Sequence,expression divergence,and complementation of homologous <Emphasis Type="Italic">ALCATRAZ</Emphasis> loci in <Emphasis Type="Italic">Brassica napus</Emphasis>

The genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus. S. Hua and I. H. Shamsi contributed equally to this work.     

Hydroxyproline O‐arabinosyltransferase mutants oppositely alter tip growth in Arabidopsis thaliana and Physcomitrella patens

Hydroxyproline O‐arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant‐specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall‐associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co‐opted to function in diverse species‐specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell‐wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall‐associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.     

Role of the gibberellin receptors GID1 during fruit‐set in Arabidopsis

Gibberellins (GAs) play a critical role in fruit‐set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit‐set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue‐specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C–RGL1 and GID1B–RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1–DELLA in the different GA‐dependent processes that occur upon fruit‐set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.     

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over‐expression induced production of more cells in the seed coat, leading to an 11–48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over‐expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over‐expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.     

Gibberellic acid signaling is required for ambient temperature‐mediated induction of flowering in Arabidopsis thaliana

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature‐dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.     

Nitrogen as a key regulator of flowering in Fagus crenata: understanding the physiological mechanism of masting by gene expression analysis

The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot‐nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.     

Selective modes determine evolutionary rates,gene compactness and expression patterns in Brassica

It has been well documented that most nuclear protein‐coding genes in organisms can be classified into two categories: positively selected genes (PSGs) and negatively selected genes (NSGs). The characteristics and evolutionary fates of different types of genes, however, have been poorly understood. In this study, the rates of nonsynonymous substitution (K_a) and the rates of synonymous substitution (K_s) were investigated by comparing the orthologs between the two sequenced Brassica species, Brassica rapa and Brassica oleracea, and the evolutionary rates, gene structures, expression patterns, and codon bias were compared between PSGs and NSGs. The resulting data show that PSGs have higher protein evolutionary rates, lower synonymous substitution rates, shorter gene length, fewer exons, higher functional specificity, lower expression level, higher tissue‐specific expression and stronger codon bias than NSGs. Although the quantities and values are different, the relative features of PSGs and NSGs have been largely verified in the model species Arabidopsis. These data suggest that PSGs and NSGs differ not only under selective pressure (K_a/K_s), but also in their evolutionary, structural and functional properties, indicating that selective modes may serve as a determinant factor for measuring evolutionary rates, gene compactness and expression patterns in Brassica.