A Genetic Dissection of Natural Variation for Stomatal Abundance Traits in Arabidopsis.

Stomatal abundance varies widely across natural populations of Arabidopsis thaliana, and presumably affects plant performance because it influences water and CO2 exchange with the atmosphere and thence photosynthesis and transpiration. In order to determine the genetic basis of this natural variation, we have analyzed a recombinant inbred line (RIL) population derived from the wild accession Ll-0 and the reference strain Landsberg erecta (Ler), which show low and high stomatal abundance, respectively. Quantitative trait locus (QTL) analyses of stomatal index, stomatal density, and pavement cell density measured in the adaxial cotyledon epidermis, identified five loci. Three of the genomic regions affect all traits and were named MID (Modulator of Cell Index and Density) 1 to 3. MID2 is a large-effect QTL overlapping with ERECTA (ER), the er-1 allele from Ler increasing all trait values. Additional analyses of natural and induced loss-of-function er mutations in different genetic backgrounds revealed that ER dysfunctions have differential and opposite effects on the stomatal index in adaxial and abaxial cotyledon epidermis and confirmed that ER is the gene underlying MID2. Ll-0 alleles at MID1 and MID3 displayed moderate and positive effects on the various traits. Furthermore, detailed developmental studies tracking primary and satellite stomatal lineages show that MID3-Ll-0 allele promotes the spacing divisions that initiate satellite lineages, while the ER allele limits them. Finally, expression analyses suggest that ER and MID3 modulate satellization through partly different regulatory pathways. Our characterization of MID3 indicates that genetic modulation of satellization contributes to the variation for stomatal abundance in natural populations, and subsequently that this trait might be involved in plant adaptation.

A Mutation in the bHLH Domain of the SPCH Transcription Factor Uncovers a BR-Dependent Mechanism for Stomatal Development.

The asymmetric cell divisions necessary for stomatal lineage initiation and progression in Arabidopsis (Arabidopsis thaliana) require the function of the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS (SPCH). Mutants lacking SPCH do not produce stomata or lineages. Here, we isolated a new spch-5 allele carrying a point mutation in the bHLH domain that displayed normal growth, but had an extremely low number of sometimes clustered stomata in the leaves, whereas the hypocotyls did not have any stomata. In vivo tracking of leaf epidermal cell divisions, combined with marker lines and genetic analysis, showed that the spch-5 leaf phenotype is dosage dependent and results from the decreased ability to initiate and amplify lineages, defects in asymmetric cell fate allocation, and misorientation of asymmetric division planes. Notably, application of brassinosteroids (BRs) partly rescued the stomatal leaf phenotype of spch-5 Transcriptomic analysis combining spch-5 with BR treatments revealed that the expression of a set of SPCH target genes was restored by BRs. Our results also show that BR-dependent stomata formation and expression of some, but not all, SPCH target genes require the integrity of the bHLH domain of SPCH.

Timely expression of the Arabidopsis stoma-fate master regulator MUTE is required for specification of other epidermal cell types.

Epidermal differentiation in Arabidopsis thaliana aerial organs involves stomatal lineage development. Lineages derive from meristemoids, which arise from asymmetric divisions of protodermal cells. Each meristemoid divides repeatedly in an inward spiral before it transits to a guard mother cell (GMC) that produces the stoma, leaving a trail of surrounding stomatal lineage ground cells (SLGCs) that eventually differentiate into endoreplicated pavement cells. MUTE is a bHLH transcription factor that is expressed in late meristemoids and drives their transition to GMCs. Loss-of-function mute mutants are stomata-less dwarf plants with arrested lineages, in which stunted putative SLGCs surround a halted meristemoid. We analysed MUTE functions using a chemically inducible system for mute-3 complementation based on conditional MUTE expression in its normal domain. Continuous induction from germination produced stomata-bearing, normal-sized plants with viable mute-3 seeds. In 2-week-old mute-3 cotyledons, meristemoids appeared to retain their identity and synchronously formed stomata in response to induced MUTE expression. However, arrested SLGCs were not complemented: many produced stomata, leading to stomatal clusters, and others remained unexpanded and diploid. In contrast, non-lineage pavement cells, which are under-endoreplicated in mute-3, expanded and increased their ploidy level upon induction, showing that the lack of response of SLGCs is specific to this arrested cell type. Leaf phenotypic mosaics include wild-type lineages and adjacent mute-3 lineages, whose meristemoids and putative SLGCs remained arrested, indicating that the role of MUTE in SLGC fate is strictly lineage-autonomous. These results show that timely MUTE expression is essential to prevent stomatal fate in SLGCs and to promote their differentiation as pavement cells.

Roles of constitutive photomorphogenic 10 in Arabidopsis stomata development.

Stomata are epidermal bi-celled structures that differentiate within special cell lineages initiated by a subset of protodermal cells. Recently, we showed that the Arabidopsis photomorphogenic repressor COP10 controls specific cell-lineage and cell-signaling developmental mechanisms in stomatal lineages. Loss-of-function cop10-1 mutant cotyledons and leaves produced (in the light and in the dark) abundant stomatal clusters, but nonlineage epidermal cells were not affected. Here we examine COP10 role in hypocotyls, cylindrical organs displaying a distinct epidermal organization with alternate files of protruding and non-protruding cells, with the latter producing a limited number of stomata. COP10 prevents stomatal clusters and restricts stomata production in hypocotyls; these roles are specific to lineage cells as in cotyledons, since COP10 loss of function does not elicit stomatal fate in nonlineage cells; COP10 also sustains the directional cell expansion of all hypocotyl epidermal cell types, and seems necessary for the differentiation between protruding and non-protruding cell files.

Dynamic analysis of epidermal cell divisions identifies specific roles for COP10 in Arabidopsis stomatal lineage development.

Stomatal development in Arabidopsis thaliana has been linked to photoreceptor-perceived light through several components of the photomorphogenic switch, whose lack of function is often seedling-lethal. CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) is an important component of this switch, its loss of function producing stomatal clusters. Exploiting the reduced lethality of the cop10-1 mutant we characterized the developmental basis of its stomatal phenotype. Constitutive, light-independent stomata overproduction accounts for half of cop10-1 stomatal abundance and appears very early in development. Clusters are responsible for the remaining stomata excess and build-up progressively at later stages. Serial impressions of living cotyledon epidermis allowed a dynamic, quantitative analysis of stomatal lineage types by reconstructing their division histories. We found that COP10 adjusts the initiation frequency and extension of stomatal lineages (entry and amplifying asymmetric divisions) and represses stomatal fate in lineage cells; COP10 also supervises the orientation of spacing divisions in satellite lineages, preventing the appearance of stomata in contact. Aberrant accumulation of the proliferating stomatal lineage cell marker TMMpro::TMM-GFP showed that the abundant cop10-1 stomatal lineages maintained extended and ectopic competence for stomatal fate. Expression of stomatal development master genes suggests that the mutant does not bypass major molecular actors in this process. cop10-1 first leaf produces trichomes and apparently normal pavement cells, but functionally and morphologically aberrant stomata; COP10 operates genetically in parallel to the stomatal repressor SDD1 and does not generally affect epidermal cell differentiation, but seems to operate on stomatal lineages where it controls specific cell-lineage and cell-signaling developmental mechanisms.

Natural variation in stomatal abundance of Arabidopsis thaliana includes cryptic diversity for different developmental processes.

BACKGROUND AND AIMS: Current understanding of stomatal development in Arabidopsis thaliana is based on mutations producing aberrant, often lethal phenotypes. The aim was to discover if naturally occurring viable phenotypes would be useful for studying stomatal development in a species that enables further molecular analysis. METHODS: Natural variation in stomatal abundance of A. thaliana was explored in two collections comprising 62 wild accessions by surveying adaxial epidermal cell-type proportion (stomatal index) and density (stomatal and pavement cell density) traits in cotyledons and first leaves. Organ size variation was studied in a subset of accessions. For all traits, maternal effects derived from different laboratory environments were evaluated. In four selected accessions, distinct stomatal initiation processes were quantitatively analysed. KEY RESULTS AND CONCLUSIONS: Substantial genetic variation was found for all six stomatal abundance-related traits, which were weakly or not affected by laboratory maternal environments. Correlation analyses revealed overall relationships among all traits. Within each organ, stomatal density highly correlated with the other traits, suggesting common genetic bases. Each trait correlated between organs, supporting supra-organ control of stomatal abundance. Clustering analyses identified accessions with uncommon phenotypic patterns, suggesting differences among genetic programmes controlling the various traits. Variation was also found in organ size, which negatively correlated with cell densities in both organs and with stomatal index in the cotyledon. Relative proportions of primary and satellite lineages varied among the accessions analysed, indicating that distinct developmental components contribute to natural diversity in stomatal abundance. Accessions with similar stomatal indices showed different lineage class ratios, revealing hidden developmental phenotypes and showing that genetic determinants of primary and satellite lineage initiation combine in several ways. This first systematic, comprehensive natural variation survey for stomatal abundance in A. thaliana reveals cryptic developmental genetic variation, and provides relevant relationships amongst stomatal traits and extreme or uncommon accessions as resources for the genetic dissection of stomatal development.

[Accumulated fluid balance in patients admitted to the ICU: is it really reliable?]. / Balance líquido acumulado en los enfermos ingresados en la UCI: ¿es realmente fiable?

In most of the textbooks, it is considered that the balance calculated after admission and the losses measured and/or estimated is an inexact way of establishing the real balance. Thus daily monitoring of the weight variations is recommended as a single possible alternative. On the other hand, there are few studies that have strictly studied the reliability of the fluid balance calculated. We also have not found any study in middle-long stay critical patients. These circumstances have led us to design an observational prospective study that will allow us to know if the accumulated balance calculated after admission and loses adequately reflect the weight changes in middle-long stay patients. We include 20 patients who were weighed every 48 hours (at least 3 times each one) and we compare the weight changes with the balances calculated. We find that, above all after the 6th day, the accumulated balance calculated adequately reflected the weight changes (mean error/day < 250 ml), regardless of the presence or not of fever, sweat, oral diet, feces or mechanical ventilation. When weight on admission to the ICU was less than 75 kg, the changes in the balance calculated adjusted even more to the weight change, the contrary occurring when the weight was greater than 75 kg. These findings suggest that the accumulated balance calculated represents a valid alternative to daily weighing of the patients and that factors such as body mass and/or surface should be taken into account to reach more exact estimations.

Balance líquido acumulado en los enfermos ingresados en la UCI: ¿es realmente fiable? / Accumulated fluid balance in patients admitted to the ICU: is it really reliable?

En la mayoría de los libros de texto se considera que el balance calculado a partir de los ingresos y las pérdidas medidas y/o estimadas, es una forma inexacta de establecer el balance real, y por ello se recomienda la monitorización diaria de las variaciones ponderales como única alternativa posible. Por otro lado, existen pocos estudios que hayan estudiado con rigor la fiabilidad del balance hídrico calculado, y además, no hemos encontrado ningún estudio en enfermos críticos de media-larga estancia. Estas circunstancias nos han motivado para diseñar un estudio prospectivo observacional que nos permitiera conocer si el balance acumulado calculado a partir de ingresos y pérdidas refleja adecuadamente los cambios ponderales en enfermos de media-larga estancia. Incluimos 20 enfermos que se pesaron cada 48 h (al menos 3 veces cada uno) y contrastamos los cambios ponderales con los balances calculados. Encontramos que, sobre todo a partir del sexto día, el balance acumulado calculado reflejó adecuadamente los cambios ponderales (error medio/día < 250 ml), independientemente de la presencia o no de fiebre, sudor, dieta oral, heces o ventilación mecánica. Cuando el peso al ingreso en UCI fue menor de 75 kg, los cambios en el balance calculado se ajustaron aún más al cambio ponderal, y sucedía lo contrario cuando el peso superaba los 75 kg. Estos hallazgos sugieren que el balance acumulado calculado representa una alternativa válida al pesaje diario de los enfermos, y que factores como la masa y/o la superficie corporal deben ser tenidos en cuenta para alcanzar estimaciones más exactas (AU)

Hipertensión pulmonar en esclerodermia / Pulmonary hypertension and scleroderma

La hipertensión arterial pulmonar (HAP) puede presentarse del 35 por ciento al 80 por ciento de los casos de esclerosis sistémica (ES), el estudio ecocardiográfico permite establecer el diagnóstico aún en pacientes asintomáticos.Objetivo: Establecer la utilidad del ecocardiograma en el diagnóstico de HAP en pacientes con ES y su probable correlación con el electrocardiograma (ECG), la radiología de tórax (Rx) y las pruebas funcionales respiratorias (PFR).Material y métodos: Se estudiaron 13 pacientes con diagnóstico de esclerosis sistémica, a todos se le realizó estudio ecocardiográfico, ECG, Rx, PFR y evaluación cardiológica. El diagnóstico de HAP se estableció con un valor superior a 25 mmHg de presión arterial pulmonar media (PAP) con ecocardigrama, al considerar como normal hasta 18 mmHg, el analisis estadístico incluyó prueba de student y X2.Resultados: El diagnóstico de HAP se estableció en el 30 por ciento de los casos considerando la PAP media ü25mmHg, el diagnóstico se incrementó hasta 54.8 por ciento; si se consideró PAP media ü18 mmHg en ambos grupos se observaron alteraciones en el ECG, Rx y PFR, sin encontrar relación estadísticamente significativa entre las variables. Clínicamente se documentó la presencia de disnea de medianos esfuerzos en el 25 por ciento y por auscultación el 30 por ciento presentó S2 pulmonar, al comparar los datos clínicos con el reporte de ECO tampoco se documentó valor estadísticamente significativo.Conclusiones: El estudio ecocardiográfico es importante en el diagnóstico y seguimiento de la HAP en pacientes con esclerodermia. No hubo correlación estadísticamente significativa con los hallazgos en el ECG, Rx y PFR y las manifestaciones clínicas encontradas.