STY1 and STY2 promote the formation of apical tissues during Arabidopsis gynoecium development

Gynoecium ontogenesis in Arabidopsis is accomplished by the co-ordinated activity of genes that control patterning and the regional differentiation of tissues, and ultimately results in the formation of a basal ovary, a short style and an apical stigma. A transposon insertion in the STYLISH1 (STY1) gene results in gynoecia with aberrant style morphology, while an insertion mutation in the closely related STYLISH2 (STY2) gene has no visible effect on gynoecium development. However, sty1-1 sty2-1 double mutant plants exhibit an enhanced sty1-1 mutant phenotype and are characterized by a further reduction in the amount of stylar and stigmatic tissues and decreased proliferation of stylar xylem. These data imply that STY1 and STY2 are partially redundant and that both genes promote style and stigma formation and influence vascular development during Arabidopsis gynoecium development. Consistently, STY1 and STY2 are expressed in the apical parts of the developing gynoecium and ectopic expression of either STY1 or STY2 driven by the CaMV 35S promoter is sufficient to transform valve cells into style cells. STY1::GUS and STY2::GUS activity is detected in many other organs as well as the gynoecium, suggesting that STY1 and STY2 may have additional functions. This is supported by the sty1-1 sty2-1 double mutants producing rosette and cauline leaves with a higher degree of serration than wild-type leaves. STY1 and STY2 are members of a small gene family, and encode proteins with a RING finger-like motif. Double mutant analyses indicate that STY1 genetically interacts with SPATULA and possibly also with CRABS CLAW.     

LEUNIG has multiple functions in gynoecium development in Arabidopsis

The Arabidopsis gene LEUNIG was previously found to regulate floral organ identity. In this work we describe gynoecial phenotypes of newly isolated strong leunig alleles, leunig-101, leunig-102, and leunig-103. Gynoecia of these strong leunig mutants are united only at the basal part, leaving four unfused parts at the apex. Among them two medial ones are styles capped with stigmas, and two lateral ones are protrusions from valves. The gynoecium with unfused apex in leunig arises as a unit from a basal meristematic zone, suggesting that LEUNIG is required for normal congenital gynoecium fusion. The epidermal cells on growing inner surfaces of leunig gynoecium failed to fuse after they contact each other, indicating that LEUNIG is essential for the proper postgenital fusion. The epidermal cells at the very distal portion of protruded valves mimic those on wild-type styles, and those valves occasionally also have stigma-like tissues, indicating that LEUNIG function is required for the valve identity determination. We have also analyzed clavata1-4 leunig-101, clavata2-1 lug-101, fruitfull-1 leunig-101, and pinoid-1 leunig-101 double mutants. clavata1-4 leunig-101 and clavata2-1 leunig-101 exhibited additive phenotypes of single mutants, suggesting that LEUNIG and CLAVATA genes function in different pathways. In contrast, FRUITFULL and PINOID genes interact with LEUNIG to regulate gynoecium development. genesis 26:42-54, 2000.     

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.     

The identification of candidate genes for a reverse genetic analysis of development and function in the Arabidopsis gynoecium

The screening for mutants and their subsequent molecular analysis has permitted the identification of a number of genes of Arabidopsis involved in the development and functions of the gynoecium. However, these processes remain far from completely understood. It is clear that in many cases, genetic redundancy and other factors can limit the efficiency of classical mutant screening. We have taken the alternative approach of a reverse genetic analysis of gene function in the Arabidopsis gynoecium. A high-throughput fluorescent differential display screen performed between two Arabidopsis floral homeotic mutants has permitted the identification of a number of genes that are specifically or preferentially expressed in the gynoecium. Here, we present the results of this screen and a detailed characterization of the expression profiles of the genes identified. Our expression analysis makes novel use of several Arabidopsis floral homeotic mutants to provide floral organ-specific gene expression profiles. The results of these studies permit the efficient targeting of effort into a functional analysis of gynoecium-expressed genes.     

The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit

The Arabidopsis fruit forms a seedpod that develops from the fertilized gynoecium. It is mainly comprised of an ovary in which three distinct tissues can be differentiated: the valves, the valve margins and the replum. Separation of cells at the valve margin allows for the valves to detach from the replum and thus dispersal of the seeds. Valves and valve margins are located in lateral positions whereas the replum is positioned medially and retains meristematic properties resembling the shoot apical meristem (SAM). Members of the WUSCHEL‐related homeobox family have been involved in stem cell maintenance in the SAM, and within this family, we found that WOX13 is expressed mainly in meristematic tissues including the replum. We also show that wox13 loss‐of‐function mutations reduce replum size and enhance the phenotypes of mutants affected in the replum identity gene RPL. Conversely, misexpression of WOX13 produces, independently from BP and RPL, an oversized replum and valve defects that closely resemble those of mutants in JAG/FIL activity genes. Our results suggest that WOX13 promotes replum development by likely preventing the activity of the JAG/FIL genes in medial tissues. This regulation seems to play a role in establishing the gradient of JAG/FIL activity along the medio‐lateral axis of the fruit critical for proper patterning. Our data have allowed us to incorporate the role of WOX13 into the regulatory network that orchestrates fruit patterning.     

Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.     

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 Are Polygalacturonases Required for Cell Separation during Reproductive Development in Arabidopsis

Cell separation is thought to involve degradation of pectin by several hydrolytic enzymes, particularly polygalacturonase (PG). Here, we characterize an activation tagging line with reduced growth and male sterility caused by increased expression of a PG encoded by QUARTET2 (QRT2). QRT2 is essential for pollen grain separation and is part of a small family of three closely related endo-PGs in the Arabidopsis thaliana proteome, including ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2. Functional assays and complementation experiments confirm that ADPG1, ADPG2, and QRT2 are PGs. Genetic analysis demonstrates that ADPG1 and ADPG2 are essential for silique dehiscence. In addition, ADPG2 and QRT2 contribute to floral organ abscission, while all three genes contribute to anther dehiscence. Expression analysis is consistent with the observed mutant phenotypes. INDEHISCENT (IND) encodes a putative basic helix-loop-helix required for silique dehiscence, and we demonstrate that the closely related HECATE3 (HEC3) gene is required for normal seed abscission and show that IND and HEC3 are required for normal expression of ADPG1 in the silique dehiscence zone and seed abscission zone, respectively. We also show that jasmonic acid and ethylene act together with abscisic acid to regulate floral organ abscission, in part by promoting QRT2 expression. These results demonstrate that multiple cell separation events, including both abscission and dehiscence, require closely related PG genes.     

Fruit indehiscence caused by enhanced expression of NO TRANSMITTING TRACT in Arabidopsis thaliana

In flowering plants, fruit dehiscence enables seed dispersal. Here we report that ntt-3D, an activation tagged allele of NO TRANSMITTING TRACT (NTT), caused a failure of fruit dehiscence in Arabidopsis. We identified ntt-3D, in which the 35S enhancer was inserted adjacent to AT3G-57670, from our activation tagged mutant library. ntt-3D mutants showed serrated leaves, short siliques, and indehiscence phenotypes. NTT-overexpressing plants largely phenocopied the ntt-3D plants. As the proximate cause of the indehiscence, ntt-3D plants exhibited a near absence of valve margin and lignified endocarp b layer in the carpel. In addition, the replum was enlarged in ntt-3D mutants. NTT expression reached a peak in flowers at stage 11 and gradually decreased thereafter and pNTT::GUS expression was mainly observed in the replum, indicating a potential role in fruit patterning. NTT:GFP localized in the nucleus and cytoplasm. FRUITFULL (FUL) expression was downregulated in ntt-3D mutants and ntt-3D suppressed upregulation of FUL in replumless mutants. These results indicate that NTT suppresses FUL, indicating a potential role in patterning of the silique. In seed crops, a reduction in pod dehiscence can increase yield by decreasing seed dispersal; therefore, our results may prove useful as a basis to improve crop yield.     

Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species

The genetic mechanisms underlying fruit development have been identified in Arabidopsis and have been comparatively studied in tomato as a representative of fleshy fruits. However, comparative expression and functional analyses on the bHLH genes downstream the genetic network, ALCATRAZ (ALC) and SPATULA (SPT), which are involved in the formation of the dehiscence zone in Arabidopsis, have not been functionally studied in the Solanaceae. Here, we perform detailed expression and functional studies of ALC/SPT homologs in Nicotiana obtusifolia with capsules, and in Capsicum annuum and Solanum lycopersicum with berries. In Solanaceae, ALC and SPT genes are expressed in leaves, and all floral organs, especially in petal margins, stamens and carpels; however, their expression changes during fruit maturation according to the fruit type. Functional analyses show that downregulation of ALC/SPT genes does not have an effect on gynoecium patterning; however, they have acquired opposite roles in petal expansion and have been co‐opted in leaf pigmentation in Solanaceae. In addition, ALC/SPT genes repress lignification in time and space during fruit development in Solanaceae. Altogether, some roles of ALC and SPT genes are different between Brassicaceae and Solanaceae; while the paralogs have undergone some subfunctionalization in the former they are mostly redundant in the latter.