SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize

The expression of class 1 knotted1-like homeobox (knox) genes affects numerous plant developmental processes, including cell-fate acquisition, lateral organ initiation, and maintenance of shoot apical meristems. The SEMAPHORE1 gene product is required for the negative regulation of a subset of maize knox genes, the duplicated loci rough sheath 1 and gnarley1 (knox4). Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots.     

Disruption of auxin transport is associated with aberrant leaf development in maize

Despite recent progress, the mechanisms governing shoot morphogenesis in higher plants are only partially understood. Classical physiological studies have suggested that gradients of the plant growth regulator auxin may play a role in controlling tissue differentiation in shoots. More recent molecular genetic studies have also identified knotted1 like homeobox (knox) genes as important regulators of shoot development. The maize (Zea mays L.) mutant rough sheath2 (rs2) displays ectopic expression of at least three knox genes and consequently conditions a range of shoot and leaf phenotypes, including aberrant vascular development, ligular displacements, and dwarfism (R. Schneeberger, M. Tsiantis, M. Freeling, J.A. Langdale [1998] Development 125: 2857–2865). In this report, we show that rs2 mutants also display decreased polar auxin transport in the shoot. We also demonstrate that germination of wild-type maize seedlings on agents known to inhibit polar auxin transport mimics aspects of the rs2 mutant phenotype. The phenotype elaborated in inhibitor-treated plants is not correlated with ectopic KNOX protein accumulation.     

Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44 degrees C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.     

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp 18.6 G2 and Ha hsp 17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp 17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25°C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42°C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp 17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.     

NARROW SHEATH1 functions from two meristematic foci during founder-cell recruitment in maize leaf development

The narrow sheath duplicate genes (ns1 and ns2) perform redundant functions during maize leaf development. Plants homozygous for mutations in both ns genes fail to develop wild-type leaf tissue in a lateral domain that includes the leaf margin. Previous studies indicated that the NS gene product(s) functions during recruitment of leaf founder-cells in a lateral, meristematic domain that contributes to leaf margin development. A mosaic analysis was performed in which the ns1-O mutation was exposed in hemizygous, clonal sectors in a genetic background already homozygous for ns2-O. Analyses of mutant, sectored plants demonstrate that NS1 function is required in L2-derived tissue layers for development of the narrow sheath leaf domain. NS1 function is not required for development of the central region of maize leaves. Furthermore, the presence of the non-mutant ns1 gene outside the narrow sheath domain cannot compensate for the absence of the non-mutant gene within the narrow sheath domain. NS1 acts non-cell autonomously within the narrow sheath-margin domain and directs recruitment of marginal, leaf founder cells from two discrete foci in the maize meristem. Loss of NS1 function during later stages of leaf development results in no phenotypic consequences. These data support our model for NS function during founder-cell recruitment in the maize meristem.     

Role of HSP101 in the stimulation of nodal root development from the coleoptilar node by light and temperature in maize (Zea mays L.) seedlings

Nodal roots (NRs) constitute the prevalent root system of adult maize plants. NRs emerge from stem nodes located below or above ground, and little is known about their inducing factors. Here, it is shown that precocious development of NRs at the coleoptilar node (NRCNs) occurred in maize seedlings when: (i) dark grown and stimulated by the concurrent action of a single light shock of low intensity white light (2 μmol m(-2) s(-1)) and a single heat shock; (ii) grown under a photoperiod of low intensity light (0.1 μmol m(-2) s(-1)); or (iii) grown in the dark under a thermoperiod (28 °C/34 °C). The light shock effects were synergistic with heat shock and with the photoperiod, whereas the thermoperiodical and photoperiodical effects were additive. Dissection of the primary root or the root cap, to mimic the fatal consequences of severe heat shock, caused negligible effects on NRCN formation, indicating that the shoot is directly involved in perception of the heat shock-inducible signal that triggered NRCN formation. A comparison between hsp101-m5::Mu1/hsp101-m5::Mu1 and Hsp101/Hsp101 seedlings indicated that the heat shock protein 101 (HSP101) chaperone inhibited NRCN formation in the light and in the dark. Stimulation of precocious NRCN formation by light and heat shocks was affected by genetic background and by the stage of seedling development. HSP101 protein levels increased in the coleoptilar node of induced wild-type plants, particularly in the procambial region, where NRCN formation originated. The adaptive relevance of development of NRCNs in response to these environmental cues and hypothetical mechanisms of regulation by HSP101 are discussed.     

Radial leaves of the maize mutant ragged seedling2 retain dorsiventral anatomy

ragged seedling2 (rgd2) is a novel, recessive mutation affecting lateral organ development in maize. The mutant phenotype of homozygous rgd2-R leaves is variable. Mild leaf phenotypes have a reduced midrib and may be moderately narrow and furcated; severe Rgd2-R(-) leaves are filamentous or even radial. Despite their radial morphology, severe Rgd2-R(-) mutant leaves develop distinct adaxial and abaxial anatomical features. Although Rgd2-R(-) mutants exhibit no reduction in adaxial or abaxial cell types, areas of epidermal cell swapping may occur that are associated with misaligned vascular bundles and outgrowths of ectopic margins. Scanning electron microscopy of young primordia and analyses of leaf developmental-marker gene expression in mutant apices reveal that RGD2 functions during recruitment of leaf founder cells and during expansive growth of leaf primordia. Overall, these phenotypes suggest that development is uncoordinated in Rgd2-R(-) mutant leaves, so that leaf components and tissues may develop quasi-independently. Models whereby RGD2 is required for developmental signaling during the initiation, anatomical patterning, and lateral expansion of maize leaves are discussed.