Of blades and branches: understanding and expanding the Arabidopsis ad/abaxial regulatory network through target gene identification.

HD-ZIPIII and KANADI transcription factors have opposing and dramatic affects on plant development. Analysis of mutants shows these proteins to be master regulators of ad/abaxial (i.e., upper/lower) leaf polarity, leaf blade outgrowth, and branch formation. Because these factors do their work by regulating other genes, we have focused our attention on defining their targets. We have found overlap between the ad/abaxial regulatory pathway and hormone signaling pathways, especially pathways of abscisic acid and auxin signaling. This has led to the discovery that abscisic acid signaling acts upstream of HD-ZIPIII and KANADI in the control of germination and may ultimately explain how environmental stress pathways control new growth at the shoot apex. Auxin signaling conversely is downstream from HD-ZIPIII and KANADI action with these factors controlling targets at all steps of auxin action-biosynthesis, transport, regulation of transport, and signaling. Based on these findings, we propose a model in which the HD-ZIPIII and KANADI factors pattern auxin response in the embryo. Finally, many genes targeted for control by HD-ZIPIII and KANADI proteins are themselves transcription factors-indicating these master regulators call up tissue specific subprograms of transcriptional control to affect the many polar differences observed across tissues.

Twenty years on: the inner workings of the shoot apical meristem, a developmental dynamo.

The shoot apical meristem of angiosperm plants generates leaf, stem and floral structures throughout the plant's lifetime. To do this, the plant must maintain a population of stem cells within the meristem while at the same time carefully controlling the placement and establishment of new leaf primordia. As there is little cell rearrangement in plants, underlying patterning mechanisms must exert careful control of cell division rates and orientations to achieve the correct final form. It has been twenty years since the first genes controlling meristem development were molecularly cloned. In the intervening decades, our understanding of the inner workings directing meristem development has increased enormously. This review summarizes our current knowledge of how the meristem functions as a persistent organ generating center. The story that emerges is one in which transcription factor activity combines with the action of the classic plant growth regulators auxin and cytokinin and with the action of more recently discovered small peptides to control proliferation and cell fate in the shoot apical meristem.

A feedback regulatory module formed by LITTLE ZIPPER and HD-ZIPIII genes.

The Arabidopsis thaliana REVOLUTA (REV) protein is a member of the class III homeodomain-leucine zipper (HD-ZIPIII) proteins. REV is a potent regulator of leaf polarity and vascular development. Here, we report the identification of a gene family that encodes small leucine zipper-containing proteins (LITTLE ZIPPER [ZPR] proteins) where the leucine zipper is similar to that found in REV, PHABULOSA, and PHAVOLUTA proteins. The transcript levels of the ZPR genes increase in response to activation of a steroid-inducible REV protein. We show that the ZPR proteins interact with REV in vitro and that ZPR3 prevents DNA binding by REV in vitro. Overexpression of ZPR proteins in Arabidopsis results in phenotypes similar to those seen when HD-ZIPIII function is reduced. We propose a negative feedback model in which REV promotes transcription of the ZPR genes. The ZPR proteins in turn form heterodimers with the REV protein, preventing it from binding DNA. The HD-ZIPIII/ZPR regulatory module would serve not only to dampen the effect of fluctuations in HD-ZIPIII protein levels but more importantly would provide a potential point of regulation (control over the ratio of inactive heterodimers to active homodimers) that could be influenced by other components of the pathway governing leaf polarity.

Giving meaning to movement.

A growing body of evidence indicates that plant transcription factors move between cells. A recent paper by Nakajima et al. (2001) shows that movement of the SHORTROOT protein provides a mechanism for signaling positional information between cell layers of the root.

Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots.

The upper side of the angiosperm leaf is specialized for efficient capture of sunlight whereas the lower side is specialized for gas exchange. In Arabidopsis, the establishment of polarity in the leaf probably requires the generation and perception of positional information along the radial (adaxial versus abaxial or central versus peripheral) dimension of the plant. This is because the future upper (adaxial) side of the leaf develops from cells closer to the centre of the shoot, whereas the future under (abaxial) side develops from cells located more peripherally. Here we implicate the Arabidopsis PHABULOSA and PHAVOLUTA genes in the perception of radial positional information in the leaf primordium. Dominant phabulosa (phb) and phavoluta (phv) mutations cause a dramatic transformation of abaxial leaf fates into adaxial leaf fates. They do so by altering the predicted sterol/lipid-binding domains of ATHB14 and ATHB9, proteins of previously unknown function that also contain DNA-binding motifs. This change probably renders the protein constitutively active, implicating this domain as a central regulator of protein function and the PHB and PHV proteins as receptors for an adaxializing signal.

Initiation of axillary and floral meristems in Arabidopsis.

Shoot development is reiterative: shoot apical meristems (SAMs) give rise to branches made of repeating leaf and stem units with new SAMs in turn formed in the axils of the leaves. Thus, new axes of growth are established on preexisting axes. Here we describe the formation of axillary meristems and floral meristems in Arabidopsis by monitoring the expression of the SHOOT MERISTEMLESS and AINTEGUMENTA genes. Expression of these genes is associated with SAMs and organ primordia, respectively. Four stages of axillary meristem development and previously undefined substages of floral meristem development are described. We find parallels between the development of axillary meristems and the development of floral meristems. Although Arabidopsis flowers develop in the apparent absence of a subtending leaf, the expression patterns of AINTEGUMENTA and SHOOT MERISTEMLESS RNAs during flower development suggest the presence of a highly reduced, "cryptic" leaf subtending the flower in Arabidopsis. We hypothesize that the STM-negative region that develops on the flanks of the inflorescence meristem is a bract primordium and that the floral meristem proper develops in the "axil" of this bract primordium. The bract primordium, although initially specified, becomes repressed in its growth.

The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene.

Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, we find that low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. We also describe defects in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, we find that embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis.

Leaf polarity and meristem formation in Arabidopsis.

Shoot apical meristems (SAMs) of seed plants are small groups of pluripotent cells responsible for making leaves, stems and flowers. While the primary SAM forms during embryogenesis, new SAMs, called axillary SAMs, develop later on the body of the plant and give rise to branches. In Arabidopsis plants, axillary SAMs develop in close association with the adaxial leaf base at the junction of the leaf and stem (the leaf axil). We describe the phenotype caused by the Arabidopsis phabulosa-1d (phb-1d) mutation. phb-1d is a dominant mutation that causes altered leaf polarity such that adaxial characters develop in place of abaxial leaf characters. The adaxialized leaves fail to develop leaf blades. This supports a recently proposed model in which the juxtaposition of ad- and abaxial cell fates is required for blade outgrowth. In addition to the alteration in leaf polarity, phb-1d mutants develop ectopic SAMs on the undersides of their leaves. Also, the phb-1d mutation weakly suppresses the shoot meristemless (stm) mutant phenotype. These observations indicate an important role for adaxial cell fate in promoting the development of axiallary SAMs and suggest a cyclical model for shoot development: SAMs make leaves which in turn are responsible for generating new SAMs.

The development of apical embryonic pattern in Arabidopsis.

The apical portion of the Arabidopsis globular stage embryo gives rise to the cotyledons and the shoot apical meristem (SAM). The SHOOT MERISTEMLESS (STM) gene is required for SAM formation during embryogenesis and for SAM function throughout the lifetime of the plant. To more precisely define the development of molecular pattern in the apical portion of the embryo, and the role of the STM gene in the development of this pattern, we have examined AINTEGUMENTA (ANT), UNUSUAL FLORAL ORGANS (UFO) and CLAVATA1 (CLV1) expression in wild-type and stm mutant embryos. The transcripts of these genes mark subdomains within the apical portion of the embryo. Our results indicate that: (1) the molecular organization characteristic of the vegetative SAM is not present in the globular embryo but instead develops gradually during embryogenesis; (2) radial pattern exists in the apical portion of the embryo prior to and independent of STM with STM expression itself responding to radial information; (3) the embryonic SAM consists of central and peripheral subdomains that express different combinations of molecular markers and differ in their ultimate fates; and (4) STM activity is required for UFO expression, STM is required for maintenance but not onset of CLV1 expression and the pattern of ANT expression is independent of STM.

Cell type specification and self renewal in the vegetative shoot apical meristem.

The vegetative shoot apical meristem of seed plants is the site of new leaf and stem formation. In the past few years genes that regulate fundamental aspects of shoot growth and development have been discovered. The recent study of these genes and their products through the use of appropriate mutants has opened new doors to understanding the molecular mechanisms of shoot apical meristem function.

Hormonal Studies of fass, an Arabidopsis Mutant That Is Altered in Organ Elongation.

We have isolated an allele of fass, an Arabidopsis thaliana mutation that separates plant development and organ differentiation from plant elongation, and studied its hormonal regulation. Micro-surgically isolated fass roots elongate 2.5 times as much as the roots on intact mutant plants. Wild-type heart embryos, when cultured with a strong auxin, naphthaleneacetic acid, phenocopy fass embryos. fass seedlings contain variable levels of free auxin, which average 2.5 times higher than wild-type seedling levels, and fass seedlings evolve 3 times as much ethylene as wild-type seedlings on a per-plant basis over a 24-h period. The length-to-width ratios of fass seedlings can be changed by several compounds that affect their endogenous ethylene levels, but fass is epistatic to etr1, an ethylene-insensitive mutant. fass's high levels of free auxin may be inducing its high levels of ethylene, which may, in turn, result in the fass phenotype. We postulate that FASS may be acting as a negative regulator to maintain wild-type auxin levels and that the mutation may be in an auxin-conjugating enzyme.

A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis.

The KNOTTED class of plant genes encodes homeodomain proteins. These genes have been found in all plant species where they have been sought and, where examined, show expression patterns that suggest they play an important role in shoot meristem function. Until now, all mutant phenotypes associated with these genes have been due to gain-of-function mutations, making it difficult to deduce their wild-type function. Here we present evidence that the Arabidopsis SHOOT-MERISTEMLESS (STM) gene, required for shoot apical meristem formation during embryogenesis, encodes a class I KNOTTED-like protein. We also describe the expression pattern of this gene in the wild-type plant. To our knowledge, STM is the first gene shown to mark a specific pattern element in the developing plant embryo both phenotypically and molecularly.

Mortality in hospitalized patients with hypoglycemia and severe hyperglycemia.

BACKGROUND: We designed a study to determine the incidence, cause, and implications of hypoglycemia (< or = 2.7 mmol/L, 49 mg/dL) and severe hyperglycemia (> or = 22.2 mmol/L, 400 mg/dL) in in-patients at an urban tertiary medical center. METHODS: A daily computer search of the Laboratory Information System identified all hospitalized patients with hypoglycemia and severe hyperglycemia during a 49-day period. Chart review was used to assess demographic information, risk factors, and epidemiologic variables. The eventual outcome of the hospitalization was obtained by follow-up. RESULTS: The incidence of hypoglycemia was 1.5%, and of hyperglycemia, 1.9%. Seventy-six percent of the hypoglycemic patients and 16% of the hyperglycemic patients had no prior history of diabetes. The mortality rate for hypoglycemic patients was 22.2%; for hyperglycemic patients it was 11.1%. For all other hospitalized patients it was 2.3% (p < 0.0001). Mortality rates for the black and Hispanic patients who were hypoglycemic (30% and 46%) were significantly higher than for white patients (6%, p < 0.01). CONCLUSIONS: Hypoglycemia and severe hyperglycemia are not uncommon in hospitalized patients and serve as metabolic markers for patients at increased risk for inhospital mortality. Early identification of at-risk patients and the impact of aggressive treatment of their underlying disease processes should be evaluated in future studies.

gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans.

We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1 (null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1 (null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells.

fog-1, a regulatory gene required for specification of spermatogenesis in the germ line of Caenorhabditis elegans.

In wild-type Caenorhabditis elegans, the XO male germ line makes only sperm and the XX hermaphrodite germ line makes sperm and then oocytes. In contrast, the germ line of either a male or a hermaphrodite carrying a mutation of the fog-1 (feminization of the germ line) locus is sexually transformed: cells that would normally make sperm differentiate as oocytes. However, the somatic tissues of fog-1 mutants remain unaffected. All fog-1 alleles identified confer the same phenotype. The fog-1 mutations appear to reduce fog-1 function, indicating that the wild-type fog-1 product is required for specification of a germ cell as a spermatocyte. Two lines of evidence indicate that a germ cell is determined for sex at about the same time that it enters meiosis. These include the fog-1 temperature sensitive period, which coincides in each sex with first entry into meiosis, and the phenotype of a fog-1; glp-1 double mutant. Experiments with double mutants show that fog-1 is epistatic to mutations in all other sex-determining genes tested. These results lead to the conclusion that fog-1 acts at the same level as the fem genes at the end of the sex determination pathway to specify germ cells as sperm.

Analysis of the role of tra-1 in germline sex determination in the nematode Caenorhabditis elegans.

In wild-type Caenorhabditis elegans there are two sexes, self-fertilizing hermaphrodites (XX) and males (XO). To investigate the role of tra-1 in controlling sex determination in germline tissue, we have examined germline phenotypes of nine tra-1 loss-of-function (lf) mutations. Previous work has shown that tra-1 is needed for female somatic development as the nongonadal soma of tra-1(lf) XX mutants is masculinized. In contrast, the germline of tra-1(lf) XX and XO animals is often feminized; a brief period of spermatogenesis is followed by oogenesis, rather than the continuous spermatogenesis observed in wild-type males. In addition, abnormal gonadal (germ line and somatic gonad) phenotypes are observed which may reflect defects in development or function of somatic gonad regulatory cells. Analysis of germline feminization and abnormal gonadal phenotypes of the various mutations alone or in trans to a deficiency reveals that they cannot be ordered in an allelic series and they do not converge to a single phenotypic endpoint. These observations lead to the suggestion that tra-1 may produce multiple products and/or is autoregulated. One interpretation of the germline feminization is that tra-1(+) is necessary for continued specification of spermatogenesis in males. We also report the isolation and characterization of tra-1 gain-of-function (gf) mutations with novel phenotypes. These include temperature sensitive, recessive germline feminization, and partial somatic loss-of-function phenotypes.

Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans.

We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.