Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

Reduced leaf complexity in tomato wiry mutants suggests a role for PHAN and KNOX genes in generating compound leaves

Recent work on species with simple leaves suggests that the juxtaposition of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf primordia is necessary for lamina outgrowth. However, how leaf dorsiventral symmetry affects leaflet formation in species with compound leaves is largely unknown. In four non-allelic dorsiventrality-defective mutants in tomato, wiry, wiry3, wiry4 and wiry6, partial or complete loss of ab-adaxiality was observed in leaves as well as in lateral organs in the flower, and the number of leaflets in leaves was reduced significantly. Morphological analyses and expression patterns of molecular markers for ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B (LeYAB B)] indicated that ab-adaxial cell fates were altered in mutant leaves. Reduction in expression of both LeT6 (a tomato KNOX gene) and LePHAN during post-primordial leaf development was correlated with a reduction in leaflet formation in the wiry mutants. LePHAN expression in LeT6 overexpression mutants suggests that LeT6 is a negative regulator of LePHAN. KNOX expression is known to be correlated with leaflet formation and we show that LeT6 requires LePHAN activity to form leaflets. These phenotypes and gene expression patterns suggest that the abaxial and adaxial domains of leaf primordia are important for leaflet primordia formation, and thus also important for compound leaf development. Furthermore, the regulatory relationship between LePHAN and KNOX genes is different from that proposed for simple-leafed species. We propose that this change in the regulatory relationship between KNOX genes and LePHAN plays a role in compound leaf development and is an important feature that distinguishes simple leaves from compound leaves.     

SIMPLE LEAF3 encodes a ribosome‐associated protein required for leaflet development in Cardamine hirsuta

Leaves show considerable variation in shape, and may be described as simple, when the leaf is entire, or dissected, when the leaf is divided into individual leaflets. Here, we report that the SIMPLE LEAF3 (SIL3) gene is a novel determinant of leaf shape in Cardamine hirsuta – a dissected‐leaved relative of the simple‐leaved model species Arabidopsis thaliana. We show that SIL3 is required for leaf growth and leaflet formation but leaf initiation is less sensitive to perturbation of SIL3 activity. SIL3 is further required for KNOX (knotted1‐like homeobox) gene expression and localized auxin activity maxima, both of which are known to promote leaflet formation. We cloned SIL3 and showed that it encodes RLI2 (RNase L inhibitor 2), an ATP binding cassette‐type ATPase with important roles in ribosome recycling and translation termination that are conserved in eukaryotes and archaea. RLI mutants have not been described in plants to date, and this paper highlights the potential of genetic studies in C. hirsuta to uncover novel gene functions. Our data indicate that leaflet development is sensitive to perturbation of RLI2‐dependent aspects of cellular growth, and link ribosome function with dissected‐leaf development.     

Auxin‐mediated lamina growth in tomato leaves is restricted by two parallel mechanisms

In the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild‐type leaf primordia, the miR160‐targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160‐targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160‐targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.     

The mutant crispa reveals multiple roles for PHANTASTICA in pea compound leaf development

Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple- and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.     

Compound leaf development and evolution in the legumes

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.     

Acropetal leaflet initiation of Eschscholzia californica is achieved by constant spacing of leaflets and differential growth of leaf

In compound leaves, leaflet primordia are initiated directionally along the lateral sides. Our understanding of the molecular basis of leaflet initiation has improved, but the regulatory mechanisms underlying spatio-temporal patterns remain unclear. In this study, we investigated the mechanisms of acropetal (from the base to the tip) progression of leaflet initiation in Eschscholzia californica. We established an ultraviolet-laser ablation system to manipulate compound-leaf development. Local ablation at the leaflet incipient site generated leaves with asymmetric morphology. In the majority of cases, leaflets that were initiated on the ablated sides shifted apically. Finite time-course observation revealed that the timing of leaflet initiation was delayed, but the distance from the leaf tip did not decrease. These results were suggestive of the local spacing mechanism in leaflet initiation, whereby the distance from the leaf tip and adjacent pre-existing leaflet determines the position of leaflet initiation. To understand how such a local patterning mechanism generates a global pattern of successive leaflet initiation, we assessed the growth rate gradient along the apical–basal axis. Our time-course analysis revealed differential growth rates along the apical–basal axis of the leaf, which can explain the acropetal progression of leaflet initiation. We propose that a leaflet is initiated at a site where the distances from pre-existing leaflets and the leaf tip are sufficient. Furthermore, the differential growth rate may be a developmental factor underlying the directionality of leaflet initiation.     

Weeds of change: Cardamine hirsuta as a new model system for studying dissected leaf development

Cardamine hirsuta, a small crucifer closely related to the model organism Arabidopsis thaliana, offers high genetic tractability and has emerged as a powerful system for studying the genetic basis for diversification of plant form. Contrary to A. thaliana, which has simple leaves, C. hirsuta produces dissected leaves divided into individual units called leaflets. Leaflet formation requires activity of Class I KNOTTED1-like homeodomain (KNOX) proteins, which also promote function of the shoot apical meristem (SAM). In C. hirsuta, KNOX genes are expressed in the leaves whereas in A. thaliana their expression is confined to the SAM, and differences in expression arise through cis-regulatory divergence of KNOX regulation. KNOX activity in C. hirsuta leaves delays the transition from proliferative growth to differentiation thus facilitating the generation of lateral growth axes that give rise to leaflets. These axes reflect the sequential generation of cell division foci across the leaf proximodistal axis in response to auxin activity maxima, which are generated by the PINFORMED1 (PIN1) auxin efflux carriers in a process that resembles organogenesis at the SAM. Delimitation of C. hirsuta leaflets also requires the activity of CUP SHAPED COTYLEDON (CUC) genes, which direct formation of organ boundaries at the SAM. These observations show how species-specific deployment of fundamental shoot development networks may have sculpted simple versus dissected leaf forms. These studies also illustrate how extending developmental genetic studies to morphologically divergent relatives of model organisms can greatly help elucidate the mechanisms underlying the evolution of form.     

CORKSCREW1 defines a novel mechanism of domain specification in the maize shoot

In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.     

Factors that affect the selection of tomato leaflets by two whiteflies,Trialeurodes vaporariorum and Bemisia tabaci (Homoptera: Aleyrodidae)

In Japan, although greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and sweet potato whitefly, Bemisia tabaci (Gennadius), co-occur on tomato plants under greenhouse conditions, the two whiteflies are distributed differently with regard to leaf position. To elucidate the factors that determine the leaf position of these whiteflies, we investigated traits for leaflets collected from three positions on tomato plants. Furthermore, we examined leaflet selection by and fertility of the two whiteflies under choice and non-choice conditions. In addition, the effect on whitefly behavior of volatile compounds released from leaflets was evaluated by use of a Y-tube olfactometer test. Nitrogen and carbon content were highest for upper leaflets. In choice tests, more T. vaporariorum and B. tabaci adults selected upper and middle leaflets, respectively. Similarly, they oviposited more eggs on upper and middle leaflets. In non-choice tests, T. vaporariorum oviposited more eggs on upper leaflets, but B. tabaci oviposited equally on each leaflet. In Y-tube olfactometer tests, more T. vaporariorum adults moved to upper leaflets whereas more B. tabaci adults moved to middle leaflets. These results suggest that different leaflet selection by adults of these two whiteflies is likely to be associated with the different volatile compounds emitted by tomato leaflets at each position.     

LEAF DEVELOPMENT IN THE NORMAL AND SOLANIFOLIA MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM)

Leaf development in the normal (lobed margin) and the solanifolia (sf/sf) mutant (entire margin) of tomato (Lycopersicon esculentum) was compared at the light and scanning electron microscope levels. The shoot apices of the mutant plants contained microbodies near the axil of the youngest leaf, which were absent in the normal plants. The structural and morphological events in the initiation of leaf primordia were similar in the two genotypes. The pattern of leaflet emergence was also similar in the two types of plants, but the timing of leaflet production was different. The first pair of leaflet primordia in the normal plants was produced on P₃, whereas in the mutant it was not produced until P₅. The adult leaves of sf/sf plants were larger than those of normal, and the greater leaf area in the mutant was associated with a greater adaxial epidermal cell and areole area. A continuous marginal fimbriate vein (MFV) was present along the margin of each of the normal leaflets. However, a continuous MFV was absent in the mutant leaflets. It is suggested that the absence of a continuous MFV in the mutant might alter the nutritional and hormonal supply to the leaf margin, which ultimately leads to a modified leaf, i.e., with an entire margin.     

Genetic networks regulated by ASYMMETRIC LEAVES1 (AS1) and AS2 in leaf development in Arabidopsis thaliana: KNOX genes control five morphological events

The asymmetric leaves 1 ( as1 ) and as2 mutants of Arabidopsis thaliana exhibit pleiotropic phenotypes. Expression of a number of genes, including three class-1 KNOTTED -like homeobox ( KNOX ) genes ( BP , KNAT2 and KNAT6 ) and ETTIN / ARF3 , is enhanced in these mutants. In the present study, we attempted to identify the phenotypic features of as1 and as2 mutants that were generated by ectopic expression of KNOX genes, using multiple loss-of-function mutations of KNOX genes as well as as1 and as2 . Our results revealed that the ectopic expression of class-1 KNOX genes resulted in reductions in the sizes of leaves, reductions in the size of sepals and petals, the formation of a less prominent midvein, the repression of adventitious root formation and late flowering. Our results also revealed that the reduction in leaf size and late flowering were caused by the repression, by KNOX genes, of a gibberellin (GA) pathway in as1 and as2 plants. The formation of a less prominent midvein and the repression of adventitious root formation were not, however, related to the GA pathway. The asymmetric formation of leaf lobes, the lower complexity of higher-ordered veins, and the elevated frequency of adventitious shoot formation on leaves of as1 and as2 plants were not rescued by multiple mutations in KNOX genes. These features must, therefore, be controlled by other genes in which expression is enhanced in the as1 and as2 mutants.     

Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines

Leaves are one of the most conspicuous and important organs of all seed plants. A fundamental source of morphological diversity in leaves is the degree to which the leaf is dissected by lobes and leaflets. We used publicly available segmental introgression lines to describe the quantitative trait loci (QTL) controlling the difference in leaf dissection seen between two tomato species, Lycopersicon esculentum and L. pennellii. We define eight morphological characteristics that comprise the mature tomato leaf and describe loci that affect each of these characters. We found 30 QTL that contribute one or more of these characters. Of these 30 QTL, 22 primarily affect leaf dissection and 8 primarily affect leaf size. On the basis of which characters are affected, four classes of loci emerge that affect leaf dissection. The majority of the QTL produce phenotypes intermediate to the two parent lines, while 5 QTL result in transgression with drastically increased dissection relative to both parent lines.     

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 ( PsGA2ox2 ) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1 -like homeobox (KNOX) genes, BP , KNAT2 and KNAT6 , which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.