Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis thaliana

The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and α-naphthalene acetic acid than others. GA(3) has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.     

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.     

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.     

Characterization of Hormonal Complex in Pea Phenotypes Differing in Leaf Morphology

Two genotypes of the pea (Pisum sativum L.) with wild-type leaves (variety Orlovchanin, Af/Af genotype) and the afila morphotype (aphyllous variety Nord, af/af genotype) were compared in terms of growth performance and hormonal characteristics of different leaf parts and the whole plant. The replacement of leaflets by tendrils in the afila variety led to a reduction in total dry weight and the area of photosynthesizing surfaces. The loss of leaflets was partly compensated for by rapid expansion of stipules at early stages of plant development and by the hypertrophy of tendrils at later stages. The excessive development of stipules in afila plants was paralleled by the increase in IAA and cytokinin level in their tissues. The hypertrophied development of tendrils and chlorophyll accumulation in tendrils of afila plants was correlated with a high IAA and cytokinin content at a low ABA background level. The elevated content of ABA in tissues of wild-type plants was associated with the preferential development of leaflets and a larger transpiratory surface compared with those in the afila form. It is assumed that this feature ensures the turgescence of wild-type plants. The possible involvement of phytohormones in growth and morphogenesis of pea mutants is discussed.     

Molecular expression of <Emphasis Type="Italic">PsPIN1</Emphasis>, a putative auxin efflux carrier gene from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

A cDNA coding for a putative auxin efflux carrier was amplified from Pisum sativum seedling shoot tips by RT-PCR and the corresponding full-length cDNA, PsPIN1, was subsequently obtained by RACE-PCR. The deduced amino acid sequence (599 residues) showed the three domain topology typical of the other PIN proteins. The PsPIN1 protein structure prediction possessed five transmembrane domains at both the N-(7-150) and C-(450-575) termini and a hydrophylic region in the middle. PsPIN1 showed highest similarity to Medicago, MtPIN4. Using the Genome Walking technique, a 1511 bp upstream region for PsPIN1 gene was sequenced. This PsPIN1 upstream region possessed multiple putative auxin, GA and light regulatory elements. The PsPIN1 mRNA was ubiquitously expressed throughout the pea plant, especially in growing tissues. Auxin induced PsPIN1 mRNA in dark grown pea seedling shoot tips. It was induced by 4-chloro-IAA, which is also an active auxin in pea, and by gibberellin (GA₃). Interestingly, the PsPIN1 mRNA was down-regulated by light treatment, possibly because light negatively regulates auxin and, especially GA levels in pea. Thus PIN1-mediated auxin efflux is a highly regulated process, not only at the level of protein localization, but also at the level of mRNA accumulation.     

Graviresponse in higher plants and its regulation in molecular bases: relevance to growth and development, and auxin polar transport in etiolated pea seedlings]

We review the graviresponse under true and simulated microgravity conditions on a clinostat in higher plants, and its regulation in molecular bases, especially on the aspect of auxin polar transport in etiolated pea (Pisum sativum L. cv. Alaska) seedlings which were the plant materials subjected to STS-95 space experiments. True and simulated microgravity conditions substantially affected growth and development in etiolated pea seedlings, especially the direction of growth of stems and roots, resulting in automorphosis. In etiolated pea seedlings grown in space, epicotyls were the most oriented toward the direction far from the cotyledons, and roots grew toward the aerial space of Plant Growth Chamber. Automorphosis observed in space were well simulated by a clinorotation on a 3-dimensional clinostat and also phenocopied by the application of auxin polar transport inhibitors of 2,3,5-triiodobenzoic acid, N-(1-naphtyl)phthalamic acid and 9-hydroxyfluorene-9-carboxylic acid. Judging from the results described above together with the fact that activities of auxin polar transport in epicotyls of etiolated pea seedlings grown in space substantially were reduced, auxin polar transport seems to be closely related to automorphosis. Strenuous efforts to learn in molecular levels how gravity contributes to the auxin polar transport in etiolated pea epicotyls resulted in successful identification of PsPIN2 and PsAUX1 genes located in plasma membrane which products are considered to be putative efflux and influx carriers of auxin, respectively. Based on the results of expression of PsPIN2 and PsAUX1 genes under various gravistimulations, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, cochleata, afila, and tendril-lessn

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.     

Expression of PIN and AUX1 genes encoding putative carrier proteins for auxin polar transport in etiolated pea epicotyls [correction of epicotyles] under simulated microgravity conditions on a three-dimensional clinostat.

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under simulated microgravity conditions on a 3-dimensional clinostat showed automorphosis-like growth and development similar to that observed in true microgravity conditions in space. Application of inhibitors of auxin polar transport phenocopied automorphosis-like growth on 1 g conditions, suggesting that automorophosis is closely related to auxin polar transport. Strenuous efforts to know the relationships between automorphosis and auxin polar transport in pea seedlings at molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin efflux and influx carrier protein, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 and AtPINs, and between PsAUX1 and AtAUX1. Expression of PsPIN1 and PsAUX1 genes in etiolated pea seedlings grown on the clinostat were substantially affected, but that of PsPIN2 was not. Roles of these genes in auxin polar transport and automorphosis of etiolated pea seedlings are also described.     

Genetic interaction and mapping studies on the leaflet development (lld) mutant in Pisum sativum

In Pisum sativum, the completely penetrant leaflet development (lld) mutation is known to sporadically abort pinnae suborgans in the unipinnate compound leaf. Here, the frequency and morphology of abortion was studied in each of the leaf suborgans in 36 genotypes and in presence of auxin and gibberellin, and their antagonists. Various lld genotypes were constructed by multifariously recombining lld with a coch homeotic stipule mutation and with af, ins, mare, mfp, tl and uni-tac leaf morphology mutations. It was observed that the suborgans at all levels of pinna subdivisions underwent lld-led abortion events at different stages of development. As in leafblades, lld aborted the pinnae in leaf-like compound coch stipules. The lld mutation interacted with mfp synergistically and with other leaf mutations additively. The rod-shaped and trumpet-shaped aborted pea leaf suborgans mimicked the phenotype of aborted leaves in HD-ZIP-III-deficient Arabidopsis thaliana mutants. Suborganwise aborted morphologies in lld gnotypes were in agreement with basipetal differentiation of leaflets and acropetal differentiation in tendrils. Altogether, the observations suggested that LLD was the master regulator of pinna development. On the basis of molecular markers found linked to lld, its locus was positioned on the linkage group III of the P. sativum genetic map.     

L-System Analysis of Compound Leaf Development in Pisum sativum L

L-system notation was used to describe mature leaf morphologyin populations of conventional, afila, tendril-less and parsley-leafpeas. Structural modules of leaves were assigned one of elevenstate symbols according to their branching potential, i.e. thenumber and arrangement of rachillae and/or tendrils or leafletsto which each would give rise after one branching iteration.State transitions at successive iterations were examined acrossgenotypes with respect to location along the leaf and node ofinsertion. Leaf branching patterns were more complex and morevariable at higher nodes. Transition outcomes decreased in complexityfrom the base to the tip of the leaf. The first transition wasthe most variable; subsequent development of the leaf was moredeterministic. Lateral appendages were more likely to branchthan central ones. Afila and tendril-less mutations increasedthe complexity of the first transition outcome over conventionalleaves. Parsley-leaf pea leaves were more complex, but lessvariable than afila leaves. Results are discussed in relationto Young's (1983) model for pea leaf morphogenesis. Pea, Pisum sativum L., L-systems, leaf, morphology, branching     

Stamina pistilloida, the Pea ortholog of Fim and UFO, is required for normal development of flowers, inflorescences, and leaves

Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2-mutations known to influence leaf, inflorescence, and flower development in pea-suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea.     

Graviresponse and its regulation from the aspect of molecular levels in higher plants: growth and development, and auxin polar transport in etiolated pea seedlings under microgravity.

In STS-95 space experiments we have demonstrated that microgravity conditions resulted in automorphosis in etiolated pea (Pisum sativum L. cv. Alaska) seedlings (Ueda et al. 1999). Automorphosis-like growth and development in etiolated pea seedlings were also induced under simulated microgravity conditions on a 3-dimensional (3-D) clinostat, epicotyls being the most oriented toward the direction far from the cotyledons. Detail analysis of epicotyl bending revealed that within 36 h after watering, no significant difference in growth direction of epicotyls was observed in between seedlings grown on the 3-D clinostat and under 1 g conditions, differential growth near the cotyledonary node resulting in epicotyl bending of ca. 45 degrees toward the direction far from the cotyledons. Thereafter epicotyls continued to grow almost straightly keeping this orientation on the 3-D clinostat. On the other hand, the growth direction in etiolated seedlings changed to antigravity direction by negative gravitropic response under 1 g conditions. Automorphological epicotyl bending was also phenocopied by the application of auxin polar transport inhibitors such as 9-hydroxyfluorene-9-carboxylic acid, N-(1-naphtyl)phthalamic acid and 2,3,5-triiodobenzoic acid. These results together with the fact that auxin polar transport activity in etiolated pea epicotyls was substantially reduced in space suggested that reduced auxin polar transport is closely related to automorphosis. Strenuous efforts to learn how gravity contributes to the auxin polar transport in etiolated pea epicotyls in molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin-efflux and influx carrier proteins, respectively. Based on the results of these gene expression under simulated microgravity conditions, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

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.     

LEAF DEVELOPMENT IN DOXANTHA UNGUIS-CATI (BIGNONIACEAE)

Leaf structure in Doxantha unguis-cati is polymorphic. The usual mature compound leaf is composed of two lanceolate leaflets and a terminal tripartite spine-tendril. Leaf primordia are initiated simultaneously in pairs on opposite flanks of the shoot apical meristem by periclinal cell divisions in the third subsurface layer of the peripheral flank meristem. Two leaflet primordia are the first lateral appendages of the compound leaf. Initiation of these leaflet primordia occurs on the adaxial side of a compound leaf primordium 63–70 μm long. Lamina formation is initiated at the base of a leaflet primordium 70–90 μm long and continues acropetally. Mesophyll differentiation occurs in later stages of development of leaflets. The second pair of lateral appendages of the leaf primordium differentiate as prongs of the tendril. Initiation of the second pair of lateral appendages occurs on the adaxial side of a primordium approximately 168 μm long. Acropetal procambialization and vacuolation of cells extend to the apex of tendrils about 112 μm long, restricting the tendril meristem to the adaxial side of the primordium and resulting in curvature of the tendril. The tendril meristem is gradually limited to a more basipetal position as elongation of apical cells continues. Initiatory divisions and early ontogenetic stages of leaflets and tendrils are similar. Their ontogeny differs when the lateral primordia are approximately 70 μm long. Marginal and submarginal initials differentiate within leaflets but not in tendrils. Apical growth of tendrils ceases very early in ontogeny as compared with leaflets.     

The place of brassinolide in the sequential response to plant growth regulators in elongating tissue

In the sequential response to plant growth regulators in young elongating tissue from peas and wheat the peak of sensitivity to 24-epi-brussinolide (1 μM) occurs after those of gibberellin and cytokinin and begins before that of auxin in isolated wheat ( Triticum vulgare L. ev. Egret) coleoptiles aged from 21-96 h. In dwarf pea ( Pisum sativum L. cv. Greenfeast) segments, the peak of sensitivity also lies between those of gibberellin and auxin, and it also occurs before sensitivity to auxin in sections from first leaves of wheat. All the leaf sections and all but the most mature coleoptiles and pea segments were sensitive to fusicocein (1 μM).