Previous studies have shown that SL promotes photomorphogenesis by increasing HY5 level . However, the molecular links from SL signaling to HY5 regulation have remained unclear. Our results show that BZS1 mediates SL regulation of HY5 level and photomorphogenesis. Similar to hy5-215, BZS1-SRDX seedlings are partially insensitive to GR24 treatment under light , which indicates that BZS1 plays a positive role in SL regulation of seedling morphogenesis. Actually, BZS1 is the only member in the subfamily IV of B-box protein family that is regulated by SL , suggesting that BZS1 plays a unique role in SL regulation of photomorphogenesis. As BZS1 increases HY5 level, SL activation of BZS1 expression would contribute, together with inactivation of COP1 , to the SL-induced HY5 accumulation. On the other hand, the BZS1-SRDX plants showed normal branching phenotypes , which suggests that BZS1 is only involved in SL regulation of HY5 activity and seedling photomorphogenesis but not shoot branching. Our finding of BZS1 function in SL response further supports a key role for BZS1 in integration of light, BR and SL signals to control seedling photomorphogenesis . To generate 15N-labeled seeds, Arabidopsis plants were grown hydroponically in diluted Hoagland solution containing 10 mM K15NO3 . One eighth diluted Hoagland medium was used at seedling stage and 1/4 Hoagland medium was used when plant started to bolt. After the siliques were fully developed, 1/8 Hoagland medium was used till seeds were fully mature. For SILIA-IP-MS assay,strawberry gutter system the 14N- or 15N-labeled seeds were grown on Hoagland medium containing 10 mM K14NO3 or K15NO3, respectively, for 5 days under constant white light.
The seedlings were harvested and ground to fine powder in liquid nitrogen. Five grams each of 14N-labeled BZS1-YFP or YFP and 15N-labeled wild-type tissue power were mixed and total proteins were extracted using extraction buffer . After removing the cell debris by centrifugation, 20 μL GFP-Trap®_MA Beads were added to the supernatant and then incubated in the cold room for 2 h with constant rotating. The beads were washed three times with IP wash buffer . The proteins were eluted twice using 50 μL 2 × SDS sample loading buffer by incubating at 95°C for 10 min. The isotope labels were switched in repeat experiments. The eluted proteins were separated by NuPAGE® Novex 4–12% Bis-Tris Gel . After Colloidal Blue staining , the gel was cut into five fractions for trypsin digestion. In-gel digestion procedure was performed according to Tang et al. . Extracted peptides were analyzed by liquid chromatographytandem mass spectrometry . The LC separation was performed using an Eksigent 425 NanoLC system on a C18 trap column and a C18 analytical column . Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile. The flow rate was 300 nL/min. The MS/MS analysis was conducted with a Thermo Scientific Q Exactive mass spectrometer in positive ion mode and data dependent acquisition mode to automatically switch between MS and MS/MS acquisition. The identification and quantification were done by pFind and pQuant softwares in an open search mode. The parameters of software were set as follows: parent mass tolerance, 15 ppm; fragment mass tolerance, 0.6 Da. The FDR of the pFind analysis was 1% for peptides. Arabidopsis TAIR10 database was used for data search. Three-day-old Arabidopsis seedlings expressing BZS1-YFP or YFP alone were grown under constant light and used for BZS1-COP1 co-immunoprecipitation assay. For the BZS1, HY5 and STH2 co-immunoprecipitation assay, about one-month-old healthy Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens GV3101 harboring corresponding plasmids.
The plants were then grown under constant light for 48 h and infiltrated leaves were collected. Total proteins from 0.3 g tissue powder were extracted with 0.6 mL extraction buffer . The lysate was pre-cleared by centrifugation twice at 20,000 g for 10 min at 4°C, and then diluted with equal volume of extraction buffer without Triton X-100. Twenty microliter of Pierce Protein A Magnetic Beads coupled with 10 μg anti-GFP polyclonal antibody were added to each protein extract and incubated at 4°C for 1 h with rotation. The beads were then collected by DynaMag™-2 Magnet and washed three times with wash buffer . The bonded proteins were eluted with 50 μL 2 × SDS loading buffer by incubating at 95°C for 10 min. For western blot analysis, proteins were separated by SDS-PAGE electrophoresis and transferred onto a nitrocellulose membrane by semi-dry transfer cell . The membrane was blocked with 5% none-fat milk followed by primary and secondary antibodies. Chemiluminescence signal was detected using SuperSignal™ West Dura Extended Duration Substrate and FluorChem™ Q System . Monoclonal GFP antibody was purchased from Clontech, USA. Myc antibody and ubiquitin antibody were from Cell Signaling Technology, USA.HY5 and COP1 antibodies were from Dr. Hongquan Yang’s lab. Secondary antibodies goat anti-mouse-HRP or goat anti-rabbitHRP were from Bio-Rad Laboratories. Arundo donax is a tall grass that is native from the lower Himalayas and invaded the Mediterranean region, prior to its introduction in the America’s . It is suspected to first have been introduced to the United States in the 1700’s, and in the Los Angeles area in the 1820’s by Spanish settlers . Its primary use was for erosion control in drainage canals.
A number of other uses for Arundo have been identified. It is the source of reeds for single reed wind instruments such as clarinet and the saxophone . In Europe and Morocco Arundo is used for waste water treatment , such as nutrient and heavy metal removal, and water volume evapotranspiration. The high rate of evapotranspiration by stands of this species, used as a benefit in these countries, is one of the characteristics that is detrimental in the California ecosystems invaded by Arundo. By the 1990’s Arundo has infested tens of thousands of acres in California riparian ecosystems, and these populations affect the functioning of these systems in different ways. It increases the fire hazard in the dry season . The regular fires promoted by the dense Arundo vegetation, are changing the nature of the ecosystem from a flood-defined to a fire-defined system . During floods, Arundo plant material can accumulate in large debris dams against flood control structures and bridges, and interfere with flood water control management , and bridges across Southern California rivers. It can grow up to 8-9 m tall, and its large leaf surface area can cause the evapotranspiration of up to 3 x the amount of water that would be lost from the water table by the native, riparian vegetation . Displacement of the native vegetation results in habitat loss for desired bird species, such as the federally endangered Least Bell’s Vireo and the threatened Willow Flycatcher . Due to the problems listed above, removal of Arundo from California ecosystems has been one of the priorities of a variety of organizations and agencies involved in the management of the state’s natural resources, such as the California Department of Fish & Game, a number of resource conservation districts. In the practice of Arundo control,grow strawberry in containers both mechanical and chemical methods of Arundo control are applied, sometimes in combination , the choice of their use depending on timing, terrain, vegetation, and funding. The risks, costs, and effects of the different control methods were listed in the most recent Arundo and saltcedar workshop by . The timing of the eradication effort can be affected by a number of factors other than the biology of the target species, such as limitations due to bird nesting season, and funding availability. Ideally, the timing of any eradication effort, chemical or mechanical should be determined by the ecophysiology of the target species, in this case Arundo donax, rather than the calendar year. For chemical eradication, this has been recognized for a while, as stated by Nelroy Jackson of Monsanto, at the first Arundo workshop: “Timing of application for optimal control is important. Best results from foliar applications of Rodeo© or Roundup© are obtained when the herbicides are applied in late summer to early fall, when the rate of downward translocation of glyphosate would be greatest.” A similar statement has not yet been made for the timing of mechanical eradication methods, nor had the effect of timing on the effectiveness of mechanical eradication been identified. Mechanical eradication of Arundo can be attempted in many different manners. The most frequently used method is the cutting of the above ground material, the plant’s tall stems. Another method of mechanical eradication is digging out the underground biomass, the rhizomes. The cutting of stems can occur before and after herbicide applications.
The large amount of standing above ground biomass, up to 45 kg/m2 impedes the removal of the cut material, because the costs will be too high. The costs associated with the removal of the large biomass of the stems, has led to the use of “chippers” that will cut the stems into pieces of approximately 5 – 10 cm in situ. After these efforts, the chipped fragments are left in place. A small fraction of the fragments left behind after chipping will contain a meristem. The stem pieces of these fragments may have been left intact, or split lengthwise. In the second case the node at which the meristem at located will have been split as well. On many pieces with a meristem, the meristem itself may still be intact. These stem fragments might sprout and regenerate into new Arundo plants . If stems are not cut into small pieces, or removed after cutting, the tall, cut stems can be washed into the watershed during a flood event. This material can accumulate behind bridges and water control structures with possible consequences as described in the introduction. Meristems on the stems can also sprout, and lead to the establishment of new stands of Arundo at the eradication project site, or down river . A. donax stands have a high stem density. The outer stalks of dense stands will start to lean to the outside because the leaves produced during the growing season push the stems in the stand apart. After the initial leaning due to crowding, gravity will pull the tall outside stems almost horizontal . Throughout this report these outside hanging stems will be referred to as “hanging stems”. The horizontal orientation causes hormonal asymmetry in these stems. The main hormones involved are IAA , GA and ethylene . The unusual IAA and GA distributions cause the side shoots developing on these hanging stems, to grow vertically. IAA also plays an important role in plant root development , and may therefore have a stimulative effect on root emergence from the adventious shoot meristem on fragments that originated from hanging stems, that would be absent in stem fragments from upright stems. In a preliminary experiment comparing root emergence between stem fragments from hanging and upright stems, 38% of the hanging stemstem fragments developed roots, while none of the upright stem-stem fragments showed root emergence . These results indicated the need for further study into the possibility that new A. donax plants can regenerate from the stem fragments with shoot meristems that might be dispersed during mechanical Arundo removal efforts. In order to apply herbicides at that time that the rate of downward translocation of photosynthates and herbicide would be greatest, this time period has to be established. Carbohydrate distribution and translocation within indeterminate plants, such as Arundo, results from the balance between the supply of carbon compounds to and the nitrogen concentration in the different plant tissues. Carbon and nitrogen are the most important elements in plant tissues. Due to different diffusion rates of NO3 – and NH4 + in soil water versus that of CO2 in air, and differences in plant N and C uptake rates, plant growth will earlier become nitrogen limited than carbon limited. During plant development tissue nitrogen concentrations are diluted by plant growth , which is mainly based on the addition of carbohydrates to the tissues. When plant growth becomes nitrogen limited, the tissue will maintain the minimum nitrogen content needed for the nucleic acids and proteins that maintain metabolic function. At this low tissue nitrogen content, there is not enough nitrogen in an individual cell to provide the nucleic acids and proteins to support the metabolism of two cells, therefore the cells cannot divide. This means that the tissue cannot grow anymore , until it receives a new supply of nitrogen.