Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae).

KEY MESSAGE: This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis. Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1 mg/L 1-Naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1 mg/L NAA and 0.1 mg/L 6-BA and that high transformation efficiency required 100 µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK application and endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

Members of the Arabidopsis FORKED1-LIKE gene family act to localize PIN1 in developing veins.

The reticulate leaf vein pattern typical of angiosperms is proposed to have been a driving force for their evolutionary success. Vein pattern is established through auxin canalization via the auxin efflux protein PINFORMED1 (PIN1). During formation of vein loops, PIN1 cellular localization is increasingly restricted to either the basal side of cells in the lower domain or to the apical side in the upper domain. We previously identified the gene FORKED1 (FKD1) to be required for PIN1 asymmetric localization and for the formation of closed vein loops. FKD1 encodes a plant-specific protein with a domain of unknown function (DUF828) and a Pleckstrin-like homology domain. The Arabidopsis genome encodes eight similar proteins, which we term the FORKED1-LIKE (FL) gene family. Five FL family members localize primarily to the trans-Golgi network or the Golgi, and several co-localize with FKD1-green flourescent protein (GFP) and RABA1c, suggesting action in the secretory pathway. While single FL gene family mutations do not result in vein pattern defects, triple mutants with mutations in FKD1, FL2, and FL3 result in a more symmetric PIN1 localization and a highly disconnected vein pattern. Our data suggest that FL genes act redundantly with FKD1 in the secretory pathway to establish appropriate PIN1 localization in provascular tissue.

Localization of Arabidopsis FORKED1 to a RABA-positive compartment suggests a role in secretion.

When FORKED1 (FKD1) is mutated, asymmetric localization of PINFORMED1 (PIN1), particularly to the apical side of cells, fails to occur properly in developing veins, resulting in an open vein pattern. FKD1 encodes a protein with a Pleckstrin homology-like (PL) domain, suggesting interaction with phosphoinositides. FKD1 has been previously found to interact with an ADP ribosylation factor GTPase-activating protein (ARF-GAP) important for vein patterning, SCARFACE/VAN3 (SFC). We find that FKD1-green fluorescent protein (GFP) localizes to the plasma membrane and to punctae labeled by SFC-yellow fluorescent protein (YFP). Supporting the idea that the FKD1 PL domain recognizes phosphatidylinositol 4-phosphate [PtdIns(4)P], FKD1-GFP co-localizes with PtdIns(4)P markers, and is more cytosolic when in a background mutant for the PtdIns(4,5)P2 hydrolases CVP2 and CVL1. Both FKD1 and SFC partially co-localize with markers for the trans-Golgi network (TGN), at which endocytic and secretory pathways merge. FKD1-labeled punctae rarely co-localize with the endocytic marker FM4-64, suggesting that FKD1 is not involved primarily in the endocytic pathway. FKD1 and SFC co-localize with members of the RABA group of RAB-GTPases, which are proposed to act in the post-Golgi secretory pathway. The compartments labeled by FKD1 and SFC do not localize to membrane compartments induced by the fungal toxin brefeldin A (BFA). Collectively, our data suggest that FKD1 and SFC act in a BFA-insensitive secretory pathway.

Water-Wisteria as an ideal plant to study heterophylly in higher aquatic plants.

KEY MESSAGE: The semi-aquatic plant Water-Wisteria is suggested as a new model to study heterophylly due to its many advantages and typical leaf phenotypic plasticity in response to environmental factors and phytohormones. Water-Wisteria, Hygrophila difformis (Acanthaceae), is a fast growing semi-aquatic plant that exhibits a variety of leaf shapes, from simple leaves to highly branched compound leaves, depending on the environment. The phenomenon by which leaves change their morphology in response to environmental conditions is called heterophylly. In order to investigate the characteristics of heterophylly, we assessed the morphology and anatomy of Hygrophila difformis in different conditions. Subsequently, we verified that phytohormones and environmental factors can induce heterophylly and found that Hygrophila difformis is easily propagated vegetatively through either leaf cuttings or callus induction, and the callus can be easily transformed by Agrobacterium tumefaciens. These results suggested that Hygrophila difformis is a good model plant to study heterophylly in higher aquatic plants.

Interventions to improve hospice and palliative care referral: a systematic review.

BACKGROUND: Hospice and palliative care are underutilized among patients at the end of their lives despite evidence that they improve patient satisfaction and reduce costs. OBJECTIVE: To synthesize evidence regarding interventions to increase hospice referral/enrollment. DESIGN AND MEASUREMENTS: We conducted a systematic review of the literature and selected studies that evaluated interventions aimed at increasing hospice use. We performed a MEDLINE search (1979 to April 2013) supplemented by manual searches of bibliographies of key articles. Study design, quality criteria, population, interventions, and outcomes for each study were extracted. The main outcome evaluated was hospice referral/enrollment. RESULTS: Our search strategy yielded 419 studies, of which only 6 met our eligibility criteria. Three studies included nursing home populations; 1 included home care patients, 1 targeted care managers, and 1 reported on heart failure patients. Three studies had a cohort design, 2 were pre-post, and only 1 was randomized. Two studies evaluated a process to identify eligible subjects. Two evaluated the impact of advance care planning programs and 2 only provided education. Interventions that only provided education showed a median increase in referral of 5% (2.8%-17%) while interventions that identified hospice candidates showed a median increase in hospice referral of 19.5 % (19%-20%). CONCLUSIONS: Interventions of different levels of complexity can improve the use of hospice services among subjects with high mortality risk. An approach that allows the medical team to assess patients' treatment goals and that engages the treating physician seems to be the most successful one.

Arabidopsis UNHINGED encodes a VPS51 homolog and reveals a role for the GARP complex in leaf shape and vein patterning.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. Directionality of PIN1 within the marginal epidermis and the presumptive veins of developing leaf primordia is crucial for establishing leaf vein pattern. One mechanism that controls PIN protein distribution within the cell membranes is endocytosis and subsequent transport to the vacuole for degradation. The Arabidopsis mutant unhinged-1 (unh-1) has simpler leaf venation with distal non-meeting of the secondary veins and fewer higher order veins, a narrower leaf with prominent serrations, and reduced root and shoot growth. We identify UNH as the Arabidopsis vacuolar protein sorting 51 (VPS51) homolog, a member of the Arabidopsis Golgi-associated retrograde protein (GARP) complex, and show that UNH interacts with VPS52, another member of the complex and colocalizes with trans Golgi network and pre-vacuolar complex markers. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the trans-Golgi network and is important in sorting proteins for lysosomal degradation. We show that vacuolar targeting is reduced in unh-1. In the epidermal cells of unh-1 leaf margins, PIN1 expression is expanded. The unh-1 leaf phenotype is partially suppressed by pin1 and cuc2-3 mutations, supporting the idea that the phenotype results from expanded PIN1 expression in the marginal epidermis. Our results suggest that UNH is important for reducing expression of PIN1 within margin cells, possibly by targeting PIN1 to the lytic vacuole.

A novel, semi-dominant allele of MONOPTEROS provides insight into leaf initiation and vein pattern formation.

Leaf vein pattern is proposed to be specified by directional auxin transport through presumptive vein cells. Activation of auxin response, which induces downstream genes that entrain auxin transport and lead to vascular differentiation, occurs through a set of transcription factors, the auxin response factors. In the absence of auxin, auxin response factors are inactive because they interact with repressor proteins, the Aux/IAA proteins. One member of the auxin response factor protein family, Auxin Response Factor 5/MONOPTEROS (MP), is critical to vein formation as indicated by reduced vein formation in loss-of-function MP alleles. We have identified a semi-dominant, gain-of-function allele of MP, autobahn or mp ( abn ), which results in vein proliferation in leaves and cotyledons. mp ( abn ) is predicted to encode a truncated product that lacks domain IV required for interaction with its Aux/IAA repressor BODENLOS (BDL). We show that the truncated product fails to interact with BDL in yeast two-hybrid assays. Ectopic expression of MP targets including the auxin efflux protein PINFORMED1 (PIN1) further supports the irrepressible nature of mp ( abn ). Asymmetric PIN1:GFP cellular localization does not occur within the enlarged PIN1:GFP expression domains, suggesting the asymmetry requires differential auxin response in neighbouring cells. Organ initiation from mp ( abn ) meristems is altered, consistent with disruption to source/sink relationships within the meristem and possible changes in gene expression. Finally, mp ( abn ) anthers fail to dehisce and their indehiscence can be relieved by jasmonic acid treatment, suggesting a specific role for MP in late anther development.

FORKED1 encodes a PH domain protein that is required for PIN1 localization in developing leaf veins.

The formation of Arabidopsis leaf veins is believed to require canalization of auxin into discrete and continuous cell files to generate a highly reproducible branched and reticulate pattern. During canalization, incipient veins become preferred routes for auxin transport through expression and asymmetric localization of the PINFORMED1 (PIN1) auxin efflux protein: PIN1 expression narrows from a group of cells to a single cell file, and localization of PIN1 protein becomes polarized to the cell membrane facing a previously formed vein. The shift in PIN1 localization is believed to require active vesicle cycling and be auxin-dependent, generating an autoregulatory loop. Previously, we have shown that fkd1 mutant leaves have an open vein pattern that lacks distal vein meeting. Here, we identify FKD1 as encoding a pleckstrin homology domain- and DUF828-containing protein. A fusion of the FKD1 promoter and the GUS reporter gene was expressed in vascular tissue throughout the plant, and its expression in incipient veins in leaves narrows in a manner similar to that of PIN1. FKD1 expression in roots and leaves can be altered by changes to auxin response and auxin transport. In the absence of FKD1, PIN1::GFP narrowing to incipient veins is delayed, and localization to the apical cell face is infrequent. The lack of apical PIN1 localization correlates with the failure of newly forming veins to connect distally with previously formed veins. Our data suggest that FKD1 influences PIN1 localization in an auxin-dependent manner, and we propose that it represents a key component of the auxin canalization pathway.

Asymmetric auxin response precedes asymmetric growth and differentiation of asymmetric leaf1 and asymmetric leaf2 Arabidopsis leaves.

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.

The FORKED genes are essential for distal vein meeting in Arabidopsis.

As in most dicotyledonous plants, the leaves and cotyledons of Arabidopsis have a closed, reticulate venation pattern. This pattern is proposed to be generated through canalization of the hormone auxin. We have identified two genes, FORKED 1 (FKD1) and FORKED 2 (FKD2), that are necessary for the closed venation pattern: mutations in either gene result in an open venation pattern that lacks distal meeting. In fkd1 leaves and cotyledons, the defect is first evident in the provascular tissue, such that the distal end of the newly forming vein does not connect to the previously formed, more distal vein. Plants doubly mutant for both genes have widespread defects in leaf venation, suggesting that the genes function in an overlapping manner at the distal junctions, but act redundantly throughout leaf veins. Expression of an auxin responsive reporter gene is reduced in fkd1 leaves, suggesting that FKD1 is necessary for the auxin response that directs vascular tissue development. The reduction in reporter gene expression and the fkd1 phenotype are relieved in the presence of auxin transport inhibition. The restoration of vein junctions in situations where auxin concentrations are increased indicates that distal vein junctions are sites of low auxin concentration and are particularly sensitive to reduced FKD1 and FKD2 activity.