Role ofLLD, a new locus for leaflet/pinna morphogenesis inPisum sativum

Properties of a mutant at theLLD (LEAF-LET DEVELOPMENT) locus in peaPisum sativum L. are reported in this paper. Plants homozygous for the Mendelian recessive mutationlld bear leaves in which a few to many leaflets are incompletely developed. Opposite pinnae of rachis nodes often formed fused incompletely developed leaflets. Thelld mutation was observed to abort pinna development at almost all morphogenetic stages. Thelld mutation demonstrated high penetrance and low expressivity. The phenotypes oflld plants intl, tac, tl tac, tl af andtl af tac backgrounds suggested that LLD function is involved in the separation of lateral adjacent blastozones differentiated on primary, secondary and tertiary rachides and lamina development in leaflets. The aborted development of tendrils and leaflets inlld mutants was related to deficiency in vascular tissue growth. The morphological and anatomical features of the leaflets formed on atl lld double mutant permitted a model of basipetal leaflet development. The key steps of leaflet morphogenesis include origin of the lamina by splitting of a radially symmetrical growing pinna having abaxial outer surface, opposite to the vascular cylinder, through an invaginational groove, differentiation of adaxial surface along the outer boundary of split tissue in the groove and expansion of the lamina ridges so formed into lamina spans.     

Organ-wise homologies of stipule, leaf and inflorescence between Pisum sativum genetic variants, Delonix regia and Caesalpinia bonduc indicate parallel evolution of morphogenetic regulation

Plants of the garden pea Pisum sativum an annual Papilionaceae species which have a mutation in the COCHLEATA (COCH) gene bear compound stipules of leaf morphology and secondary inflorescences in which flowers are borne in axils of bracteoles on an inflorescence stem. In the wild-type P. sativum stipules are simple foliaceous and flowers are non-bracteolated. Mutants of the coch phenotype are not known in any other plant species. The COCH gene of P. sativum is not yet sequenced. Therefore whether or not the COCH gene is present in species other than P. sativum remains unknown. Based on the principle of parallel evolution, it is thought that there may be leguminous species that possess the coch phenotype. In search of coch species, the Leguminosae flora of Delhi was surveyed for species that demonstrate compound stipules and bracteolated inflorescences. Out of 124 reported in the Leguminosae flora of Delhi, only the perennial Caesalpinioideae tree species Delonix regia and the shrubby vine species Caesalpinia bonduc were observed to have features of the coch mutants of P. sativum. Since the traits of simple foliaceous stipules and bracteoleless secondary inflorescences of wild-type P. sativum are common to many species among more than 19,400 species of family Leguminosae, it is hypothesized that the COCH gene may be as common as the ubiquitously present LEAFY gene (orthologue of UNIFOLIATA gene of P. sativum) in angiosperms.     

Parallelismic homoplasy of leaf and stipule phenotypes among genetic variants of Pisum sativum and Medicago truncatula and some taxa of Papilionoideae, Caesalpinioideae and Mimosoideae subfamilies of the Leguminosae flora of Delhi

The leguminous flora of Delhi comprises 78 Papilionoideae, 24 Caesalpinioideae and 24 Mimosoideae species; 80 of them are perennials. Five types of imparipinnate and two types of paripinnate compound leaves were observed in the species. The paripinnate leaves are bipinnate in 25 species (mostly mimosoid) and bifoliate in two species. The imparipinnate leaves were trifoliate or multifoliate in 59 papilionoid species and multifoliate in 16 caesalpinioid species; four of the papilionoid species produced leafletted and tendrilled unipinnate leaves. Leaves were bifacially simple in 22 species, simple with ectopic terminal growth in one species and simple tendril in one species. Twenty-one species (mostly mimosoid) were devoid of stipules. In 82 species stipules were small and free. Stipules were large and lobed in 17 species and large and adnate in four species. Two species of Caesalpinioideae produce compound leaf-like stipules. All four stipule phenotypes of 126 species corresponded with stipular phenotypes observed in wild type, coch, st and coch st genotypes of the model legume P. sativum. The seven leaf phenotypes observed in 126 species corresponded with phenotypes expected among combinations of uni (uni-tac), af, ins, mfp and tl mutants of P. sativum and sgl1, cfl1, slm1 and palm1 mutants of M. truncatula, also an IRL model legume. All the variation in leaf and stipule morphologies observed in the leguminous flora of Delhi could be explained in terms of the gene regulatory networks already revealed in P. sativum and M. truncatula. It is hypothesized that the ancestral gene regulatory networks for leaves and stipules produced in Leguminosae were like that prevalent in P. sativum.     

Leaf and Flower Development in Pea (Pisum sativum L.): Mutants cochleata andunifoliata

The stipule mutant cochleata(coch) and the simple-leaf mutantunifoliata(uni) are utilized to increase understanding of the controlof compound leaf and flower development in pea. The phenotypeof the coch mutant, which affects the basal stipules of thepea leaf, is described in detail. Mutant coch flowers have supernumeraryorgans, abnormal fusing of flower parts, mosaic organs and partialmale and female sterility. The wild-type Coch gene is shownto have a role in inflorescence development, floral organ identityand in the positioning of leaf parts. Changes in meristem sizemay be related to changes in leaf morphology. In the coch mutant,stipule primordia are small and their development is retardedin comparison with that of the first leaflet primordia. Thediameter of the shoot apical meristem of the uni mutant is approx.25% less than that of its wild-type siblings. This is the firsttime that a significant difference in apical meristem size hasbeen observed in a pea leaf mutant. Genetic controls in thebasal part of the leaf are illustrated by interactions betweencoch and other mutants. The mutantcoch gene is shown to changestipules into a more ‘compound leaf-like’ identitywhich is not affected by thestipules reduced mutation. The interactionof coch and tendril-less(tl) genes reveals that the expressionof the wild-type Tl gene is reduced at the base of the leaf,supporting the theories of gradients of gene action. Copyright2001 Annals of Botany Company Pisum sativum, garden pea, leaf morphogenesis, compound leaf, leaf mutants, flower morphology     

Mapping of the <Emphasis Type="Italic">multifoliate pinna</Emphasis> (<Emphasis Type="Italic">mfp</Emphasis>) leaf-blade morphology mutation in grain pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

The multifoliate pinna (mfp) mutation alters the leaf-blade architecture of pea, such that simple tendril pinnae of distal domain are replaced by compound pinna blades of tendrilled leaflets in mfp homozygotes. The MFP locus was mapped with reference to DNA markers using F₂ and F_2:5 RIL as mapping populations. Among 205 RAPD, 27 ISSR and 35 SSR markers that demonstrated polymorphism between the parents of mapping populations, three RAPD markers were found linked to the MFP locus by bulk segregant analyses on mfp/mfp and MFP/MFP bulks assembled from the F_2:5 population. The segregational analysis of mfp and 267 DNA markers on 96 F₂ plants allowed placement of 26 DNA markers with reference to MFP on a linkage group. The existence of common markers on reference genetic maps and MFP linkage group developed here showed that MFP is located on linkage group IV of the consensus genetic map of pea.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Auxin–cytokinin and auxin–gibberellin interactions during morphogenesis of the compound leaves of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

INDETERMINATE GROWTH OF LEAVES IN GUAREA (MELIACEAE): A TWIG ANALOGUE

Leaves of seed plants are generally characterized as organs of determinate growth. In this regard, Guarea and related genera seem unusual in that the pinnately compound leaves of these plants contain a bud at their tip from which new pinnae expand from time to time. Previous studies (based upon superficial examinations of leaf-tip buds) have produced contradictory conclusions regarding how long the leaf apex remains meristematic and produces new pinna primordia. In order to determine whether leaf development in Guarea is truly indeterminate, we microscopically examined leaf-tip buds of G. guidonia and G. glabra. In both species, the leaf apex remains meristematic and continues to produce new pinna primordia as the leaf ages. Unexpanded leaves of G. guidonia contained an average of 23 pinna primordia, while the oldest leaves we examined had initiated an average of 44 total pinnae. In G. glabra, unexpanded leaves contained 8 pinnae, whereas an average of 28 pinnae had been initiated on the oldest leaves. These results indicate that leaf development in Guarea is truly indeterminate. Periodic examination of individual intact leaves indicated that the leaves commonly continue their growth for 2 or more years (observed maximum = 51 months). As new leaflets are initiated at the shoot apex (and subsequently expand in rhythmic flushes), older (basal) leaflets may abscise. In addition, the petiole and rachis of the leaf thicken and become woody as a result of the activity of a vascular cambium. Guarea leaves therefore seem to function as the analogue of a typical twig (stem) in general habit as well as in their indeterminate apical growth and secondary thickening.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.     

Developmental anatomy of the three-dimensional leaf ofBotrychium ternatum (Thunb.) Sw.

The ophioglossaceous leaf exhibits a unique morphology, three-dimensional constitution. This examination clarified the developmental manner of the leaf ofBotrychium ternatum (Thunb.) Sw. The leaf primordium develops as an ordinary appendicular leaf which shows a typical hyponastic growth curvature, though it soon takes a conical shape with a tetrahedral apical cell. The leaf primordium forms a sporophyll primordium first, then forms vegetative primary pinnae acting as the trophophyll primordium. The sporophyll primordium also forms sporogenous primary pinnae. The sporophyll initiation begins with establishment of a new apical cell (sporophyll apical cell) near the original leaf apical cell. Through activity of the sporophyll apical cell most of the sporophyll primordium is formed later. In contrast, the vegetative or sporogenous primary pinna begins to develop as a low mound of surface cells on the trophophyll or sporophyll primordium respectively. The apical cell is later established on the summit of each pinna primordium. In the developmental point of view, the sporophyll primordium is not equivalent to the primary pinnae on the trophophyll primordium. The sporophyll may not represent two fused basal pinnae of a leaf, but represents an organ independent of and equivalent to the trophophyll.     

The determination of pea leaves,leaflets, and tendrils

Pea leaf determination was examined by culturing excised leaf, leaflet, and tendril primordia of different ages on a nutrient medium. Pinna primordia were designated as 1) determined, if they grew normally in culture; 2) undetermined, if they grew into differentiated structures that were morphologically and anatomically different from either leaflet or tendril; or 3) partially determined, if the two pinnae of an opposite pair developed unequally in isolation, or for leaflet pinnae only, if laminae were initiated but did not develop completely. The compound pea leaf as a whole is determined over four plastochrons of development. Proximal pinnae are determined during the second leaf plastochron, approximately 0.8 plastochron after their initiation. The second most proximal pair of pinnae is determined during the third plastochron, and the terminal portion of the rachis is determined last, during the fourth plastochron. Determination of leaflet dorsiventrality is gradual, requiring a critical minimum period with the leaf in physiological contact with the shoot system. The rachis primordium, when isolated from the shoot, does not affect determination of its pinnae as leaflets or tendrils. Afila and tendril-less homeotic mutations do not alter the timing of pinna determination.     

The reduced folate carrier-1 (RFC1 696T>C) polymorphism is associated with spontaneously aborted embryos in Koreans

The major cause of early spontaneous abortion is believed to be chromosomal abnormality. However, the genetic etiologies of spontaneous abortion are still unknown. A central feature of fetal development is widespread rapid cell division. Due to its role in DNA synthesis, the need for folate increases during periods of rapid fetal growth. Folate transport across cell membranes is mediated by reduced folate carrier-1 (RFC1). Variants within SLC19A1 may influence folate and homocysteine concentrations. The aim of this study was to investigate the association of RFC1 mutations with spontaneous abortion in aborted embryos. We studied 115 spontaneously aborted embryos at <20 weeks of gestational age, 102 child controls, and 353 adult controls. The genotype frequencies of RFC1 polymorphisms, 80A>G and 696T>C, in spontaneously aborted embryos were measured. The RFC1 696T>C mutation was significantly increased in spontaneously aborted embryos compared to child controls. Further studies will be required to examine the functional significance of the RFC1 696T>C polymorphism.     

Roles of the af and tl genes in pea leaf morphogenesis: characterization of the double mutant (afaftltl)

The pleiofila phenotype (afaftltl double mutant) of Pisum sativum arises from two single-gene, recessive mutations known to affect the identity of leaf pinnae, afila (af), and acacia (tl). The wild-type leaf consists of proximal leaflets and distal tendrils, whereas the pleiofila leaf consists of branched pinnae terminating in small leaflets. Using morphological measurements, histology, and SEM, we characterized the variation in leaf form along the plant axis, in leaflet anatomy, and in leaf development in embryonic, early postembryonic, and late postembryonic leaves of aftl and wild-type plants. Leaves on aftl plants increase in complexity more rapidly during shoot ontogeny than those on wild-type plants. Leaflets of aftl plants have identical histology to wild-type leaflets although they have smaller and fewer cells. Pinna initiation is acropetal in early postembryonic leaves of aftl plants and in all leaves of wild-type plants, whereas in late postembryonic leaves of aftl plants pinna initiation is bidirectional. Most phenotypic differences between these genotypes can be attributed to differential timing (heterochrony) of major developmental events.     

INDETERMINATE GROWTH AND RAMIFICATION OF THE CLIMBING LEAVES OF LYGODIUM JAPONICUM (SCHIZAEACEAE)

Morphological and anatomical specializations of the climbing leaves (CL) of Lygodium japonicum were investigated. Examination of growth relationships between the rachis and pinnae of the circumnutating CL revealed a close relationship to the “searcher” morphology of twining shoots. The CL has resting pinna apices (leafbuds) capable of replacing a damaged leaf apex or ramifying the foliar axis. Their structure and growth is similar to the main leaf apex. CL growth is indeterminate and occurs at a steady rate. Crozier uncoiling and rachis elongation occurs by a mechanism of unequal rates of cell division and elongation. The adaptations of the CL are interpreted as specializations within the basic principles of fern leaf morphogenesis.     

Pea Compound Leaf Architecture Is Regulated by Interactions among the Genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea.     

Genetic analyses of the formation of the serrated margin of leaf blades in Arabidopsis : combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules

Developmental control of the formation of the serrated margin of leaf blades was investigated. First, the expression was characterized of a marker gene encoding β-glucuronidase in strain #1-35-38, a transgenic strain of Arabidopsis thaliana (L.) Heynh, derived by the use of a previously described transposon-tagging system. In strain #1-35-38, expression of the marker gene was tissue-specific, being restricted to stipules and the toothed margins of laminae. Using this transgenic marker gene, we examined the development of leaf blade margins in Arabidopsis. We compared the pattern of expression of the marker gene in the leaves of the wild-type plant with that in plants carrying the asymmetric leaves1 (as1) mutation, which causes dramatic changes in leaf-blade morphology in Arabidopsis. The as1 mutant showed normal morphology of early leaf primordia. The mutation affected the development of leaf segmentation in Arabidopsis without any change in the number or morphology of cells in laminae. The as1 mutation affected leaf morphology independently of mutations in other genes known to affect leaf morphogenesis, such as the acaulis1 mutation and the angustifolia mutation. Based upon these results, the development of the morphology of leaf margins in Arabidopsis is discussed.     

TheAfGene Regulates Timing and Direction of Major Developmental Events during Leaf Morphogenesis in Garden Pea (Pisum sativum)

The wildtype leaf of the garden pea possesses proximal pairsof leaflets and distal pairs of tendrils in the blade region.Theafila (af) mutation causes leaflets to be replaced by compound(branched) tendrils. We characterized the morphological variationin leaf form along the plant axis and leaf development in earlyand late postembryonic leaves onafilaplants to infer the roleof theAfgene. Leaf forms are more diverse early in shoot ontogenyonafilaplants.Afinfluences pinna length and pinna branchingin addition to pinna type. Pinna initiation in the proximalregion ofafilaleaf primordia is basipetal and delayed comparedto wildtype plants. In addition, pinna development in the proximalregion ofafilaleaves occurs for a longer period of time thanon wildtype leaf primordia. Therefore,Afregulates the timingand direction of leaf developmental processes in the proximalregion of the leaf, but has little effect on the distal region.These data support the heterochronic model of pea leaf morphogenesisproposed by Luet al. (International Journal of Plant Science157:311–355, 1996).Copyright 1999 Annals of Botany Company. afila,Fabaceae, garden pea, heterochrony, leaf morphogenesis,Pisum sativum.