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Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.

Nuclear proteins are primarily regulatory factors governing gene expression. Multiple factors determine the localization of a protein in the nucleus. An upright identification of nuclear proteins is way far from accuracy. We have attempted to combine information from subcellular prediction tools, experimental evidence, and nuclear proteome data to identify a reliable list of seed-expressed nuclear proteins in rice. Depending upon the number of prediction tools calling a protein nuclear, we could sort 19,441 seed expressed proteins into five categories. Of which, half of the seed-expressed proteins were called nuclear by at least one out of four prediction tools. Further, gene ontology (GO) enrichment and transcription factor composition analysis showed that 6116 seed-expressed proteins could be called nuclear with a greater assertion. Localization evidence from experimental data was available for 1360 proteins. Their analysis showed that a 92.04% accuracy of a nuclear call is valid for proteins predicted nuclear by at least three tools. Distribution of nuclear localization signals and nuclear export signals showed that the majority of category four members were nuclear resident proteins, whereas other categories have a low fraction of nuclear resident proteins and significantly higher constitution of shuttling proteins. We compiled all the above information for the seed-expressed genes in the form of a searchable database named Rice Seed Nuclear Protein DataBase (RSNP-DB) https://pmb.du.ac.in/rsnpdb . This information will be useful for comprehending the role of seed nuclear proteome in rice.

Flooding due to extreme weather threatens crops and ecosystems. To understand variation in gene regulatory networks activated by submergence, we conducted a high-resolution analysis of chromatin accessibility and gene expression at three scales of transcript control in four angiosperms, ranging from a dryland-adapted wild species to a wetland crop. The data define a cohort of conserved submergence-activated genes with signatures of overlapping cis regulation by four transcription factor families. Syntenic genes are more highly expressed than nonsyntenic genes, yet both can have the cis motifs and chromatin accessibility associated with submergence up-regulation. Whereas the flexible circuitry spans the eudicot-monocot divide, the frequency of specific cis motifs, extent of chromatin accessibility, and degree of submergence activation are more prevalent in the wetland crop and may have adaptive importance., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plants show leaf form alteration in response to changes in the surrounding environment, and this phenomenon is called heterophylly. Although heterophylly is seen across plant species, the regulatory mechanisms involved are largely unknown. Here, we investigated the mechanism underlying heterophylly in Rorippa aquatica (Brassicaceae), also known as North American lake cress. R. aquatica develops pinnately dissected leaves in submerged conditions, whereas it forms simple leaves with serrated margins in terrestrial conditions. We found that the expression levels of KNOTTED1-LIKE HOMEOBOX (KNOX1) orthologs changed in response to changes in the surrounding environment (e.g., change of ambient temperature; below or above water) and that the accumulation of gibberellin (GA), which is thought to be regulated by KNOX1 genes, also changed in the leaf primordia. We further demonstrated that exogenous GA affects the complexity of leaf form in this species. Moreover, RNA-seq revealed a relationship between light intensity and leaf form. These results suggest that regulation of GA level via KNOX1 genes is involved in regulating heterophylly in R. aquatica. The mechanism responsible for morphological diversification of leaf form among species may also govern the variation of leaf form within a species in response to environmental changes., (© 2014 American Society of Plant Biologists. All rights reserved.)

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Class I KNOTTED1-LIKE HOMEOBOX (KNOX1) genes are expressed in the shoot apical meristem (SAM) to effect its formation and maintenance. KNOX1 genes are also involved in leaf shape control throughout angiosperm evolution. Leaves can be classified as either simple or compound, and KNOX1 expression patterns in leaf primordia are highly correlated with leaf shape; in most simple-leafed species, KNOX1 genes are expressed only in the SAM but not in leaf primordia, while in compound-leafed species they are expressed both in the SAM and leaf primordia. How can KNOX1 expression be maintained to a high degree in the SAM, but simultaneously be so variable in leaves? This dichotomy suggests that the processes of leaf and SAM development have been compartmentalized during evolution. Here, we introduce our findings regarding the regulation of expression of SHOOT MERISTEMLESS, a KNOX1 gene, together with a brief review of KNOX1 genes from an evolutionary viewpoint. We also present our findings regarding another aspect of KNOX1 regulation via a protein-protein interaction network involved in the natural variation in leaf shape. Both aspects of KNOX1 regulation could be utilized for fine-tuning leaf morphology during evolution without affecting the essential function of KNOX genes in the shoot.

Striking diversity in size, arrangement, and complexity of leaves can sometimes be seen in closely related species. One such variation is found between wild tomato species collected by Charles Darwin from the Galapagos Islands [1-5]. Here, we show that a single-nucleotide deletion in the promoter of the PETROSELINUM (PTS) [3] gene upregulates the gene product in leaves and is responsible for the natural variation in leaf shape in the Galapagean tomatoes. PTS encodes a novel KNOTTED1-LIKE HOMEOBOX (KNOX) gene that lacks a homeodomain. We also showed that the tomato classical mutant bipinnata (bip) [6], which recapitulates the Pts phenotype, results from the loss of function of a BEL-LIKE HOMEODOMAIN (BELL) gene, BIP. We used bimolecular fluorescence complementation and two-hybrid competition assays to show that PTS represses KNOX1 protein interactions with BIP, as well as subsequent nuclear localization of this transcriptional complex. We suggest that natural variation in leaf shape can be created with a rheostat-like mechanism that alters the KNOX1 protein interaction network specifically during leaf development. This subtle change in interaction between transcription factors leaves essential KNOX1 function in the shoot apical meristem intact and appears to be a facile way to alter leaf morphology during evolution.

The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX (KNOX1) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in Arabidopsis (hereafter called STM genes) in the initiating primordia. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5' upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 (AS1/2) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes.

Plant development requires regulation of both cell division and differentiation. The class 1 KNOTTED1-like homeobox (KNOX) genes such as knotted1 (kn1) in maize (Zea mays) and SHOOTMERISTEMLESS in Arabidopsis (Arabidopsis thaliana) play a role in maintaining shoot apical meristem indeterminacy, and their misexpression is sufficient to induce cell division and meristem formation. KNOX overexpression experiments have shown that these genes interact with the cytokinin, auxin, and gibberellin pathways. The L1 layer has been shown to be necessary for the maintenance of indeterminacy in the underlying meristem layers. This work explores the possibility that the L1 affects meristem function by disrupting hormone transport pathways. The semidominant Extra cell layers1 (Xcl1) mutation in maize leads to the production of multiple epidermal layers by overproduction of a normal gene product. Meristem size is reduced in mutant plants and more cells are incorporated into the incipient leaf primordium. Thus, Xcl1 may provide a link between L1 division patterns, hormonal pathways, and meristem maintenance. We used double mutants between Xcl1 and dominant KNOX mutants and showed that Xcl1 suppresses the Kn1 phenotype but has a synergistic interaction with gnarley1 and rough sheath1, possibly correlated with changes in gibberellin and auxin signaling. In addition, double mutants between Xcl1 and crinkly4 had defects in shoot meristem maintenance. Thus, proper L1 development is essential for meristem function, and XCL1 may act to coordinate hormonal effects with KNOX gene function at the shoot apex.

Knotted-like homeobox (KNOX) genes encode important regulators of shoot development in flowering plants. In Arabidopsis, class I KNOX genes are part of a regulatory system that contributes to indeterminacy of shoot development, delimitation of leaf primordia and internode development. In other species, class I KNOX genes have also been recruited in the control of marginal blastozone fractionation during dissected leaf development. Here we report the isolation of class I KNOX genes from two species of the basal eudicot family Papaveraceae, Chelidonium majus and Eschscholzia californica. Sequence comparisons and expression patterns indicate that these genes are orthologs of SHOOTMERISTEMLESS (STM), a class I KNOX gene from Arabidopsis. Both genes are expressed in the center of vegetative and floral shoot apical meristems (SAM), but downregulated at leaf or floral organ initiating sites. While Eschscholzia californica STM (EcSTM) is again upregulated during acropetal pinna formation, in situ hybridization could not detect Chelidonium majus STM (CmSTM) transcripts at any stage of basipetal leaf development, indicating divergent evolution of STM gene function in leaves within Papaveraceae. Immunolocalization of KNOX proteins indicate that other gene family members may control leaf dissection in both species. The contrasting direction of pinna initiation in the two species was also investigated using Histone H4 expression. Leaves at early stages of development did not reveal notable differences in cell division activity of the elongating leaf axis, suggesting that differential meristematic growth may not play a role in determining the observed dissection patterns.

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.

Diverse leaf forms in nature can be categorized as simple or compound. Simple leaves, such as those of petunia, have a single unit of blade, whereas compound leaves, such as those of tomato, have several units of blades called leaflets. Compound leaves can be pinnate, with leaflets arranged in succession on a rachis, or palmate, with leaflets clustered together at the leaf tip. The mechanisms that generate these various leaf forms are largely unknown. The upper (adaxial) surface is usually different from the bottom (abaxial) surface in both simple and compound leaves. In species with simple leaves, the specification of adaxial and abaxial cells is important for formation of the leaf blade, and the MYB transcription factor gene PHANTASTICA (PHAN) is involved in maintaining the leaf adaxial (upper) domain. Here we show that downregulation of PHAN is sufficient to reduce the adaxial domain of leaf primordia and to change pinnate compound leaves into palmate compound leaves. Furthermore, this mechanism seems to be shared among compound leaves that arose independently.

Differentiation of plant cells is regulated by position-dependent mechanisms rather than lineage. The maize Extra cell layers1 (Xcl1) mutation causes oblique, periclinal divisions to occur in the protoderm layer. These protodermal periclinal divisions occur at the expense of normal anticlinal divisions and cause the production of extra cell layers with epidermal characteristics, indicating that cells are differentiating according to lineage instead of position. Mutant kernels have several aleurone layers instead of one, indicating that Xcl1 alters cell division orientation in cells that divide predominantly in the anticlinal plane. Dosage analysis of Xcl1 reveals that the mutant phenotype is caused by overproduction of a normal gene product. This allows cells that have already received differentiation signals to continue to divide in aberrant planes and suggests that the timing of cell division determines differentiation. Cells that divide early and in the absence of differentiation signals use positional information, while cells that divide late after perceiving differentiation signals use lineage information instead of position.

The Sucrose export defective1 (Sxd1) gene of maize was cloned and shown to encode a novel protein conserved between plants and cyanobacteria. The structure of the Sxd1 locus was determined in wild-type plants and two independent sxd1 alleles. Expression analysis demonstrated that the gene was transcribed in all green tissues, with highest levels in maturing leaf blades. In situ hybridization studies revealed high levels of Sxd1 mRNA in bundle sheath cells, with lower levels within the mesophyll. The SXD1 protein was localized to chloroplasts, in both bundle sheath and mesophyll cells. Levels of sucrose, glucose, and fructose were compared between wild-type and sxd1 plants. Mutant plants were fully capable of producing sucrose and accumulated all three sugars at concentrations above those measured in wild-type plants. Despite these increased sugar concentrations, photosynthetic gene expression was not significantly downregulated in affected areas of sxd1 leaf blades. These results are consistent with photosynthate being trapped within anthocyanin-accumulating regions of sxd1 leaves due to plasmodesmal occlusion at the bundle sheath-vascular parenchyma boundary of the minor veins. A model for SXD1 function is proposed in which the protein is involved in a chloroplast-to-nucleus signaling pathway necessary for proper late-stage differentiation of maize bundle sheath cells, including the developmentally regulated modification of plasmodesmata.

Knotted-like (KNOX) proteins constitute a group of homeodomain proteins involved in pattern formation in developing tissues of angiosperms and other green plants. We conducted phylogenetic analyses of nucleotide and amino acid sequences of all known KNOX proteins in order to examine their evolution. Our analyses reveal two groups of KNOX proteins, classes I and II. Dicot and monocot sequences occur in both classes, indicating that the protein classes arose prior to the origin of the monocots. A conifer (Picea) sequence is nested within class I, suggesting that there are likely to be other copies of KNOX genes in this and other conifers. The orthology of several grass genes (including Zea Kn1, ZMKN1) is strongly supported by phylogenetic and synteny analyses. However, no compelling evidence supports the hypothesis of orthology previously proposed for several dicot genes and ZMKN1. Analysis of expression patterns suggests that the ancestral KNOX gene was expressed in all plant parts and that the propensity to be downregulated in roots and leaves evolved in the class I genes.

Homeobox genes are known to play a role in developmental regulation. The knotted-like homeobox (knox) genes fall into two classes. The class I knox genes like knl, stml, and knatl are involved in maintaining meristem identity in cells. The function of class II knox genes is at yet undetermined. We have characterized two knox genes from tomato. LeT6 and LeT12 map to distinct chromosome locations that are different from the location for a recently cloned knox gene from tomato, tknl, confirming that plant homeobox genes are not clustered on chromosomes. These genes have a distinct expression pattern. Unlike other class I knl-like genes, LeT6 is expressed in developing lateral organs and developing ovaries in flowers. LeT12 is more ubiquitously expressed in the mature plant. RNA in situ localization data suggest that both these genes may have a role to play in formative events in ovule and embryo morphogenesis.

The KNOTTED-1 (KN1) locus of maize is defined by dominant mutations that affect leaf cell fates. Transposon tagging led to the isolation of the gene and the discovery that KN1 encodes a homeo domain. Immunolocalization studies showed that in wild-type maize plants, KN1 protein is present in nuclei of apical meristems and immature shoot axes but is down-regulated as lateral organs, such as leaves, are initiated. The protein is not immunohistochemically detectable in wild-type leaves at any stage. In developing leaves of plants carrying the dominant Kn1 mutation, temporally and spatially restricted ectopic expression of KN1 causes the mutant phenotype. To better understand the function of KN1 in plant development, we sought to determine the phenotype of plants in which KN1 was constitutively expressed. We find that tobacco plants transformed with the KN1 cDNA driven by the CaMV 35S promoter have a dramatically altered phenotype. The phenotypes are variable and depend on the level of KN1 protein. Plants expressing moderate levels of KN1 are reduced in stature with rumpled or lobed leaves. Plants with relatively high levels of KN1 lack apical dominance and are severely dwarfed in overall height and leaf size. Small shoots originate from the surface of these diminutive leaves. On the basis of phenotypes in maize and tobacco, we propose that the KN1 homeo box gene plays a role in determining cell fate. The consequences of KN1 overexpression appear to depend on the concentration of KN1 and the timing of its expression during organogenesis.