SPATULA and ALCATRAZ, are partially redundant, functionally diverging bHLH genes required for Arabidopsis gynoecium and fruit development

The Arabidopsis gynoecium is a complex organ that facilitates fertilization, later developing into a dehiscent silique that protects seeds until their dispersal. Identifying genes important for development is often hampered by functional redundancy. We report unequal redundancy between two closely related genes, SPATULA (SPT) and ALCATRAZ (ALC), revealing previously unknown developmental roles for each. SPT is known to support septum, style and stigma development in the flower, whereas ALC is involved in dehiscence zone development in the fruit. ALC diverged from a SPT-like ancestor following gene duplication coinciding with the At-β polyploidy event. Here we show that ALC is also involved in early gynoecium development, and SPT in later valve margin generation in the silique. Evidence includes the increased severity of early gynoecium disruption, and of later valve margin defects, in spt-alc double mutants. In addition, a repressive version of SPT (35S:SPT-SRDX) disrupts both structures. Consistent with redundancy, ALC and SPT expression patterns overlap in these tissues, and the ALC promoter carries two atypical E-box elements identical to one in SPT required for valve margin expression. Further, SPT can heterodimerize with ALC, and 35S:SPT can fully complement dehiscence defects in alc mutants, although 35S:ALC can only partly complement spt gynoecium disruptions, perhaps associated with its sequence simplification. Interactions with FRUITFULL and SHATTERPROOF genes differ somewhat between SPT and ALC, reflecting their different specializations. These two genes are apparently undergoing subfunctionalization, with SPT essential for earlier carpel margin tissues, and ALC specializing in later dehiscence zone development.     

CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS

To help understand the process of carpel morphogenesis, the roles of three carpel development genes have been partitioned genetically. Mutants of CRABS CLAW cause the gynoecium to develop into a wider but shorter structure, and the two carpels are unfused at the apex. Mutants of a second gene, SPATULA, show reduced growth of the style, stigma, and septum, and the transmitting tract is absent. Double mutants of crabs claw and spatula with homeotic mutants that develop ectopic carpels demonstrate that CRABS CLAW and SPATULA are necessary for, and inseparable from, carpel development, and that their action is negatively regulated by A and B organ identity genes. The third carpel gene studied, AGAMOUS, encodes C function that has been proposed to fully specify carpel identity. When AGAMOUS function is removed together with the A class gene APETALA2, however, the organs retain many carpelloid properties, suggesting that other genes are also involved. We show here that further mutant disruption of both CRABS CLAW and SPATULA function removes remaining carpelloid properties, revealing that the three genes together are necessary to generate the mature gynoecium. In particular, AGAMOUS is required to specify the identity of the carpel wall and to promote the stylar outgrowth at the apex, CRABS CLAW suppresses radial growth of the developing gynoecium but promotes its longitudinal growth, and SPATULA supports development of the carpel margins and tissues derived from them. The three genes mostly act independently, although there is genetic evidence that CRABS CLAW enhances AGAMOUS and SPATULA function.     

Involvement of CUP-SHAPED COTYLEDON genes in gynoecium and ovule development in Arabidopsis thaliana

When mutations in CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 are combined, severe defects involving fusion of sepals and of stamens occur in Arabidopsis flowers. In addition, septa of gynoecia do not fuse along the length of the ovaries and many ovules have their growth arrested. CUC2 is expressed at the tips of septal primordia during gynoecium development and at the boundary between nucellus and chalaza during ovule development. These expression patterns are partially consistent with the phenotype of the mutant gynoecium. CUC2 mRNA is also shown to be expressed at the boundaries between meristems and organ primordia during both the vegetative and reproductive phases. This expression pattern indicates that CUC2 is generally involved in organ separation in shoot and floral meristems.     

Regulation of gynoecium marginal tissue formation by LEUNIG and AINTEGUMENTA

The carpel is the female reproductive organ of flowering plants. In Arabidopsis, congenital fusion of two carpels leads to the formation of an enclosed gynoecium. The margins of the two fused carpels are meristematic in nature and give rise to placentas, ovules, septa, abaxial repla, and the majority of the stylar and stigmatic tissues. Thus, understanding how the marginal tissues are specified and identifying genes that direct their development may provide important insight into higher plant reproductive development. In this study, we show that LEUNIG and AINTEGUMENTA are two critical regulators of marginal tissue development. Double mutants of leunig aintegumenta fail to develop placentas, ovules, septa, stigma, and style. This effect is specific to the leunig aintegumenta double mutant and is not found in other double mutant combinations such as leunig apetala2 or aintegumenta apetala2. Additional analyses indicate that the absence of marginal tissues in leunig aintegumenta double mutants is not mediated by ectopic AGAMOUS. We propose that LEUNIG and AINTEGUMENTA act together to control the expression of common target genes that regulate cell proliferation associated with marginal tissue development.     

Expression of PaNAC01, a Picea abies CUP-SHAPED COTYLEDON orthologue, is regulated by polar auxin transport and associated with differentiation of the shoot apical meristem and formation of separated cotyledons

Background and Aims During embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana, the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC-like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT. Methods Sixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant. Key Results Two full-length CUC-like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant. Conclusions The results show that CUC-like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.     

The CUP-SHAPED COTYLEDON genes promote adventitious shoot formation on calli

In Arabidopsis, shoots regenerate on calli derived from hypocotyl explants. Mutations in CUC1 and CUC2 (CUP-SHAPED COTYLEDON) reduce the induction of adventitious shoots on calli. To elucidate the function of CUC1 and CUC2 during this process, these genes were overexpressed in calli. Our results indicate that CUC1 and CUC2 promote adventitious shoot formation on calli. To clarify their functions, the concentrations of auxin and cytokinin in the shoot-inducing medium were changed. Calli of the single and double mutants of cuc1 and cuc2, as well as calli overexpressing either of the CUC genes, responded similarly. This suggests that neither of the genes are involved in synthesis or sensitivity of these hormones. During embryogenesis, CUC1 and CUC2 induce shoot apical meristem formation through activation of STM (SHOOT MERISTEMLESS). Our analyses using the stm mutant and an STM::GUS construct suggest that CUC1 and CUC2 also function upstream of STM even in calli.     

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.     

The Carpel of Moringa

The gynoecium of Moringa is tricarpellary, syncarpous and unilocularwith parietal placentation. The three carpel primordia ariseindependently but soon become connected resulting in an annularstructure which develops into the tubular gynoecium. The gynoeciumis supplied with three dorsal and three marginal bundles. Thelatter represent the fusion product of the marginal bundlesof adjacent carpels and each splits into three in the ovarywall. The ovules receive their vascular supply from a commonbundle, which branches from the dorsal trace of the carpel atthe base of the ovary. The derivation of the gynoecium fromconduplicate carpels is postulated. Moringa oleifera, carpel morphology, conduplicate carpel, carpel ontogeny     

Flower and fruit characters in the early-divergent lamiid family Metteniusaceae, with particular reference to the evolution of pseudomonomery

Evaluating the morphological relationships of angiosperm families that still remain unplaced in the current systems of classification is challenging because it requires comparative data across a broad phylogenetic range. The small neotropical family Metteniusaceae was recently placed within the lamiids, as sister to either the enigmatic Oncothecaceae or the clade (Boraginaceae + Gentianales + Lamiales + Solanales + Vahliaceae). We examined the development of two of the primary diagnostic traits of Metteniusaceae, the moniliform anthers and the unilocular gynoecium. The gynoecium is 5-carpellate, and contains two ovules with a massive, vascularized integument. Late sympetaly and unitegmic ovules support placement of Metteniusaceae in the lamiids. The 5-carpellate gynoecium is consistent with a sister-group relationship between Metteniusaceae and Oncothecaceae. The gynoecium of Metteniusaceae is unusual in that it is monosymmetric throughout ontogeny, which indicates pseudomonomery; the five carpel initials are congenitally fused by their margins and form a single locule; the two ovules develop from the two smallest and most poorly developed lateral carpels. Comparisons with other pseudomonomerous taxa allow us to propose division of the complex processes leading to pseudomonomery into eight characters, including carpel number and fusion, gynoecial symmetry, timing of carpel reduction, and number and position of nonfertile carpels.