Phenotypic characterization of the CRISPA (ARP gene) mutant of pea (Pisum sativum; Fabaceae): a reevaluation.

PREMISE OF THE STUDY: Leaf form and development are controlled genetically. The ARP genes encode MYB transcription factors that interact with Class 1 KNOX genes in a regulatory module that controls meristem-leaf determinations and is highly conserved in plants. ARP loss of function alleles and subsequent KNOX1 overexpression cause many unusual leaf phenotypes including loss or partial loss of the ability to produce a lamina and production of "knots" on leaf blades. CRISPA (CRI) is the ARP gene in pea, and a number of its mutant alleles are known. METHODS: We made morphological and anatomical evaluations of cri-1 mutant plants while controlling for genetic background and for heteroblastic effects, and we used aldehyde fixation and resin preparations for anatomical analysis. Further, we compared gene expression in WT and cri-1 shoot tips and HOP1/PsKN1 and CRI expression in other leaf mutants. KEY RESULTS: The cri-1 plants had more extensive abnormalities in the proximal than in the distal regions of the leaf, including ectopic stipules, narrow leaflets, and shortened petioles with excessive adaxial expansion. "Knots" were morphologically and anatomically variable but consisted of vascularized out-pocketing of the adaxial leaflet surface. HOP1/PsKN1 and UNI mRNA levels were higher in cri-1 shoot tips, and some auxin-regulated genes were lower. Low LE expression suggests that the GA level is high in cri-1 shoot tips. CONCLUSIONS: The CRISPA gene of pea suppresses KNOX1 genes and UNI and functions to (1) maintain proximal-distal regions in their appropriate positions, (2) restrict excessive adaxial cell proliferation, and (3) promote laminar expansion.

Unifoliata-Afila interactions in pea leaf morphogenesis.

PREMISE OF THE STUDY: Processes of leaf morphogenesis provide the basis for the great diversity of leaf form among higher plants. The common garden pea (Pisum sativum) offers a developmental model system for understanding how gene and hormone interactions impart a large array of mutant leaf phenotypes. • METHODS: To understand the role of auxin in AF and UNI gene function and their interaction, we compared the range of leaf phenotypes on afila (af) and unifoliata (uni) double mutants, examined the effects of these mutations on auxin levels, auxin transport, auxin response via DR5::GUS, and expression of auxin-regulated genes. • KEY RESULTS: The adult leaves of af uni double mutants have leaflets and tendrils and typically possess two lateral pinna pairs and a terminal leaflet. The af mutants have higher auxin content, stronger auxin response, and higher expression of auxin responsive genes than wildtype. The uni mutant has reduced auxin content and transport, whereas the uni-tac mutant has higher auxin content and transport and reduced auxin response compared to wildtype. • CONCLUSIONS: Auxin concentration and response differences characterize the antagonistic relationship between AF and UNI in pea leaf development. The mechanism involves modulation of auxin mediated by one or both genes; UNI is expressed in and promotes high auxin levels, and AF suppresses auxin levels.

Interactions between GA, auxin, and UNI expression controlling shoot ontogeny, leaf morphogenesis, and auxin response in Pisum sativum (Fabaceae): or how the uni-tac mutant is rescued.

PREMISE OF THE STUDY: Leaf morphogenesis, including that of compound leaves, provides the basis for the great diversity of leaf form among higher plants. Leaf form is an important character by which plants adapt to their environment. The common garden pea provides a developmental model system for understanding leaf development in the legumes and a contrasting one for other groups of plants. METHODS: We used genetic, tissue culture, and physiological methods, as well as DR5::GUS expression and qRT-PCR, to explore the interactions between the hormones gibberellic acid (GA) and auxin and Unifoliata ( UNI ) gene expression that control leaf morphogenesis in pea. KEY RESULTS: Rate of increase in leaf complexity during shoot ontogeny (i.e., heteroblasty) and adult leaf complexity are controlled by GA through UNI . Leaves on greenhouse-grown uni-tac mutants are rescued by weekly GA or auxin applications. Auxin responsiveness is reduced in uni-tac shoot and root tips and in wild-type shoot tips treated with auxin transport inhibitors. GA and auxin increase UNI mRNA levels in uni-tac as well as that of other transcription factors. CONCLUSIONS: GA and auxin positively promote leaf dissection during leaf morphogenesis in pea by prolonging the time during which acropetally initiated pinna pairs are produced. GA-generated elaboration of leaf morphogenesis is in distinct contrast to that in other species, such as tomato and Cardamine . Instead, GA and auxin play common and supportive roles in pea leaf morphogenesis as they do in many other aspects of plant development

Dosage effect of the short arm of chromosome 1 of rye on root morphology and anatomy in bread wheat.

The spontaneous translocation of the short arm of chromosome 1 of rye (1RS) in bread wheat is associated with higher root biomass and grain yield. Recent studies have confirmed the presence of QTL for different root morphological traits on the 1RS arm in bread wheat. This study was conducted to address two questions in wheat root genetics. First, does the presence of the 1RS arm in bread wheat affect its root anatomy? Second, how does root morphology and anatomy of bread wheat respond to different dosages of 1RS? Near-isogenic plants with a different number (0 to 4 dosages) of 1RS translocations were studied for root morphology and anatomy. The F(1) hybrid, with single doses of the 1RS and 1AS arms, showed heterosis for root and shoot biomass. In other genotypes, with 0, 2, or 4 doses of 1RS, root biomass was incremental with the increase in the dosage of 1RS in bread wheat. This study also provided evidence of the presence of gene(s) influencing root xylem vessel number, size, and distribution in bread wheat. It was found that root vasculature follows a specific developmental pattern along the length of the tap root and 1RS dosage tends to affect the transitions differentially in different positions. This study indicated that the inherent differences in root morphology and anatomy of different 1RS lines may be advantageous compared to normal bread wheat to survive under stress conditions.

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation.

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites with a broad host range which includes economically important crop species. Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume grown in many regions where root-knot nematodes are a major problem in production fields. Several sources of resistance to root-knot nematode have been identified in cowpea, including the widely used Rk gene. As part of a study to elucidate the mechanism of Rk-mediated resistance, the histological response to avirulent M. incognita feeding of a resistant cowpea cultivar CB46 was compared with a susceptible near-isogenic line (in CB46 background). Most root-knot nematode resistance mechanisms in host plants that have been examined induced a hypersensitive response (HR). However, there was no typical HR in resistant cowpea roots and nematodes were able to develop normal feeding sites similar to those in susceptible roots up to 9-14 d post inoculation (dpi). From 14-21 dpi giant cell deterioration was observed and the female nematodes showed arrested development and deterioration. Nematodes failed to reach maturity and did not initiate egg laying in resistant roots. These results confirmed that the induction of resistance is relatively late in this system. Typically in pathogen resistance HR is closely associated with an oxidative burst (OB) in infected tissue. The level of reactive oxygen species release in both compatible and incompatible reactions during early and late stages of infection was also quantified. Following a basal OB during early infection in both susceptible and resistant roots, which was also observed in mechanically wounded root tissues, no significant OB was detected up to 14 dpi, a profile consistent with the histological observations of a delayed resistance response. These results will be useful to design gene expression experiments to dissect Rk-mediated resistance at the molecular level.

Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.

Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Synchronized coexpression of wild-type BMV and FHV genome components in plant cells resulted in the assembly of virions packaging the respective viral progeny RNA. To further elucidate the link between replication and packaging, coat protein (CP) open reading frames were precisely exchanged between BMV RNA 3 (B3) and FHV RNA 2 (F2), creating chimeric RNAs expressing heterologous CP genes (B3/FCP and F2/BCP). Coinfiltration of each chimera with its corresponding genome counterpart to provide viral replicase (B1+B2+B3/FCP and F1+F2/BCP) resulted in the expected progeny profiles, but virions exhibited a nonspecific packaging phenotype. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.

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.

Genetic effects on the biomass partitioning and growth of Pisum and Lycopersicon.

We examined a series of eight pea genotypes differing in three naturally occurring allelic mutations, i.e., af (afila), st (stipules reduced), and tl (tendril-less) and three species, five cultivars, and one interspecific hybrid of tomato differing in SP (SELF-PRUNING) allele composition to determine whether different phenotypes ontogenetically express different biomass partitioning patterns compared to the isometric partitioning pattern and an interspecific 3/4 scaling "rule" governing annual growth with respect to body mass. The slopes and "elevations" (i.e., α and log ß, respectively) of log-log linear regression curves of bivariate plots of leaf, stem, and root dry mass and of annual growth vs. total body mass were used to assess pattern homogeneity. The annual growth of all pea and tomato phenotypes complied with the 3/4 growth rule. The biomass partitioning patterns of all tomato phenotypes were statistically indistinguishable from the isometric pattern as were those of the pea wild type and three single-mutant genotypes. However, significant departures from the isometric (and pea wild type) biomass allocation pattern were observed for three genotypes, all of which were homozygous for the af allele. These results open the door to explore the heritability and genetics underlying the allometry of biomass partitioning patterns and growth.

Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips.

The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development.

Auxin-cytokinin and auxin-gibberellin interactions during morphogenesis of the compound leaves of pea (Pisum sativum).

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.

Roles for auxin during morphogenesis of the compound leaves of pea ( Pisum sativum).

The goal of this study was to explore the impact of the plant growth regulator auxin on the development of compound leaves in pea. Wildtype ( WT) plantlets, as well as those of two leaf mutants, acacia ( tl) and tendrilled acacia ( uni-tac) of pea ( Pisum sativum L.), were grown on media containing the auxin-transport inhibitors 2,3,5-triiodobenzoic acid (TIBA), N-(1-naphthyl)phthalamic acid (NPA), or the auxin antagonist, p-chlorophenoxyisobutyric acid (PCIB). The resulting plantlets were carefully analyzed morphologically, by scanning electron microscopy and for Uni gene expression using quantitative reverse transcription-polymerase chain reaction. Auxin transport was measured in WT leaf parts using [(14)C]indole-3-acetic acid. Relative Uni gene expression was determined in shoot tips of a range of leaf-form mutants. Morphological abnormalities were observed for all genotypes examined. The terminal tendrils on WT plants were converted to leaflets, stubs or were aborted. The number of pinna pairs produced on leaves was reduced, with the distal forms being eliminated before the proximal ones. Some leaves were converted to simple, including tri-and bilobed, forms. These treatments phenocopy the uni-tac and unifoliata ( uni) mutants of pea. In the most extreme situations, leaf blades were completely lost leaving only a pair of stipules or scale leaves. Polar auxin transport was basipetal for all leaf parts. Uni gene expression in shoot tips was significantly reduced in 60 microM NPA and TIBA. Uni mRNA was more abundant in tl, af and af tl and reduced in the uni mutants compared to WT. These results indicate that an auxin gradient plays fundamental roles in controlling morphogenesis in the compound leaves of pea and specifically it: (i). is the driving force for leaf growth and pinna determination; (ii). is necessary for pinna initiation; and (iii). controls subsequent pinna development.

DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS.

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.