Defying gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and are a resource for understanding how plants regulate posture control. The peach (Prunus persica) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Little is known about the function of WEEP despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach trees do not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster lateral root gravitropic response. This suggests that WEEP moderates root gravitropism and is essential to establishing the set-point angle of lateral roots from the gravity vector. Additionally, size exclusion chromatography indicated that WEEP proteins self-oligomerize, like other proteins with sterile alpha motif domains. Collectively, our results from weeping peach provide insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Defying Gravity: WEEP promotes negative gravitropism in Prunus persica (peach) shoots and roots by establishing asymmetric auxin gradients.

Trees with weeping shoot architectures are valued for their beauty and serve as tremendous resources for understanding how plants regulate posture control. The Prunus persica (peach) weeping phenotype, which has elliptical downward arching branches, is caused by a homozygous mutation in the WEEP gene. Until now, little was known about the function of WEEP protein despite its high conservation throughout Plantae. Here, we present the results of anatomical, biochemical, biomechanical, physiological, and molecular experiments that provide insight into WEEP function. Our data suggest that weeping peach does not have defects in branch structure. Rather, transcriptomes from the adaxial (upper) and abaxial (lower) sides of standard and weeping branch shoot tips revealed flipped expression patterns for genes associated with early auxin response, tissue patterning, cell elongation, and tension wood development. This suggests that WEEP promotes polar auxin transport toward the lower side during shoot gravitropic response, leading to cell elongation and tension wood development. In addition, weeping peach trees exhibited steeper root systems and faster root gravitropic response, just as barley and wheat with mutations in their WEEP homolog EGT2. This suggests that the role of WEEP in regulating lateral organ angles and orientations during gravitropism may be conserved. Additionally, size-exclusion chromatography indicated that WEEP proteins self-oligomerize, like other SAM-domain proteins. This oligomerization may be required for WEEP to function in formation of protein complexes during auxin transport. Collectively, our results from weeping peach provide new insight into polar auxin transport mechanisms associated with gravitropism and lateral shoot and root orientation.

Opposing influences of TAC1 and LAZY1 on Lateral Shoot Orientation in Arabidopsis.

TAC1 and LAZY1 are members of a gene family that regulates lateral shoot orientation in plants. TAC1 promotes outward orientations in response to light, while LAZY1 promotes upward shoot orientations in response to gravity via altered auxin transport. We performed genetic, molecular, and biochemical assays to investigate possible interactions between these genes. In Arabidopsis they were expressed in similar tissues and double mutants revealed the wide-angled lazy1 branch phenotype, indicating it is epistatic to the tac1 shoot phenotype. Surprisingly, the lack of TAC1 did not influence gravitropic shoot curvature responses. Combined, these results suggest TAC1 might negatively regulate LAZY1 to promote outward shoot orientations. However, additional results revealed that TAC1- and LAZY1 influence on shoot orientation is more complex than a simple direct negative regulatory pathway. Transcriptomes of Arabidopsis tac1 and lazy1 mutants compared to wild type under normal and gravistimulated conditions revealed few overlapping differentially expressed genes. Overexpression of each gene did not result in major branch angle differences. Shoot tip hormone levels were similar between tac1, lazy1, and Col, apart from exceptionally elevated levels of salicylic acid in lazy1. The data presented here provide a foundation for future study of TAC1 and LAZY1 regulation of shoot architecture.

Branching out: new insights into the genetic regulation of shoot architecture in trees.

Directional growth in all plants involves both phototropic and gravitropic responses. Accordingly, mechanisms controlling shoot architecture throughout the plant kingdom are likely similar. However, as forms vary between species due in part to gene copy number and functional divergence, some aspects of how plants predetermine and regulate architecture can differ. This is especially true when comparing annual herbaceous species (e.g. model plants) to woody perennials such as trees. In the past decade, inexpensive genomic sequencing and technological advances enabled gene discovery and functional analyses in trees. This led to the identification of genes associated with tree shoot architecture control. Here, we present recent discoveries on the regulation of shoot architectures for which causative genes have been identified, including dwarf, weeping, columnar, and pillar growth habits. We also discuss potential applications of these findings.

Patterned Deposition of Xylan and Lignin is Independent from that of the Secondary Wall Cellulose of Arabidopsis Xylem Vessels.

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7bac , despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7mur10-2 , and an established knock-out mutant, cesa7irx3-4 Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane.

Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

The Arabidopsis cellulose synthase complex: a proposed hexamer of CESA trimers in an equimolar stoichiometry.

Cellulose is the most abundant renewable polymer on Earth and a major component of the plant cell wall. In vascular plants, cellulose synthesis is catalyzed by a large, plasma membrane-localized cellulose synthase complex (CSC), visualized as a hexameric rosette structure. Three unique cellulose synthase (CESA) isoforms are required for CSC assembly and function. However, elucidation of either the number or stoichiometry of CESAs within the CSC has remained elusive. In this study, we show a 1:1:1 stoichiometry between the three Arabidopsis thaliana secondary cell wall isozymes: CESA4, CESA7, and CESA8. This ratio was determined utilizing a simple but elegant method of quantitative immunoblotting using isoform-specific antibodies and (35)S-labeled protein standards for each CESA. Additionally, the observed equimolar stoichiometry was found to be fixed along the axis of the stem, which represents a developmental gradient. Our results complement recent spectroscopic analyses pointing toward an 18-chain cellulose microfibril. Taken together, we propose that the CSC is composed of a hexamer of catalytically active CESA trimers, with each CESA in equimolar amounts. This finding is a crucial advance in understanding how CESAs integrate to form higher order complexes, which is a key determinate of cellulose microfibril and cell wall properties.

Comparison of sterile versus nonsterile acellular dermal matrices for breast reconstruction.

BACKGROUND: Acellular dermal matrix (ADM) has been associated with an increased incidence of complications after implant-based breast reconstruction. Recently, sterile ADM has been introduced in an attempt to minimize these complications. To analyze the impact of this product on patient outcomes, we created a database of patients undergoing implant-based breast reconstruction. METHODS: Patients undergoing implant-based breast reconstruction at the University of Kentucky Medical Center from January 1, 2011, to December 31, 2011 were identified. A database of patient characteristics and outcomes was created. Outcomes investigated included mastectomy flap necrosis, dehiscence, infection, red breast, capsular contracture, hematoma, and seroma. Statistical analysis was performed. RESULTS: Fifty-eight patients underwent breast reconstruction with implants or tissue expanders. Of the 58 patients, 9 had the sterile form of ADM placed, 25 had the original aseptic but not sterile ADM, and 24 were not reconstructed with ADM. The most frequent complication noted was seroma, occurring in 6/9 patients with sterile ADM as compared to 2/25 with the aseptic ADM. This was statistically significant (P = 0.003). CONCLUSIONS: The use of sterile ADM is associated with a statistically significant increase in seroma formation. The etiology of this increased incidence remains unknown, but it correlates with the introduction of the sterile form of ADM at our institution. A different preparation or sterilization process, or some other variable as yet unknown, may be responsible. Further studies comparing the different forms of ADM in an animal model may serve to clarify this issue.

Cranialization in a cohort of 154 consecutive patients with frontal sinus fractures (1987-2007): review and update of a compelling procedure in the selected patient.

Retrospective review of charts of 180 consecutive patients with frontal sinus fractures managed by plastic surgeons at the University of Kentucky between 1987 and 2007 was performed with institutional review board approval. Twenty-six charts did not meet the criteria. The remaining 154 records provided 1-to-20-year follow-up. The study included 34 patients who underwent cranialization and 120 patients who did not. A low-complication rate of 6% after cranialization is ascribed by the authors to meticulous sinus mucosal debridement; thorough obliteration of the frontal sinus outflow tract (with sterile gelatin sponge pledgets and bone chips from the outer cortex of the temporoparietal skull); and avoidance of avascular barriers, such as abdominal fat. As high-resolution computerized tomography with parasaggital views was introduced, an increasing ability to preoperatively define the extent of injury of the medial and lateral sinus floor was observed. The authors conclude selective use of cranialization is indicated.

Infectious complications associated with the use of acellular dermal matrix in implant-based bilateral breast reconstruction.

BACKGROUND: The use of acellular dermal matrix (ADM) has become a routine practice in implant-based breast reconstruction. Bilateral mastectomy is becoming more popular in cases of unilateral breast cancer. ADM has been associated with an increased incidence of complications. METHODS: We identified cases of bilateral implant-based breast reconstruction over a 5-year period. Data collection included medical comorbities, details of operative management, and details of postoperative cancer treatment. RESULTS: On univariate analysis, the use of ADM (31% vs. 7%, P = 0.018), smoking (37% vs. 13%, P = 0.045), and open wound (55% vs. 13%, P = 0.006) were significantly associated with increased risk of infection. Multivariate analysis revealed open wound as the strongest predictor of infection. CONCLUSIONS: The use of ADM is associated with an increased risk of infection in bilateral implant-based breast reconstruction. However, it does not appear to be an independent risk factor by itself.

Microsurgical reconstruction of large, locally advanced cutaneous malignancy of the head and neck.

Large, locally advanced cutaneous malignancy of the head and neck region is rare. However, when present, they impart a significant reconstructive challenge. These cancers have a tendency to invade peripheral tissues covering a large surface area as well as expose deeper structures such as skull, dura, orbit, and sinus after resection. Complicating the reconstructive dilemma is the high incidence of individuals who have undergone previous surgery in the region as well as adjuvant radiation therapy, which may preclude the use of local flaps or skin graft. Free tissue transfer provides a reconstructive surgeon the ability to provide well-vascularized tissue with adequate volume not limited by arc of rotation.