Journal of Bioinformatics and Proteomics Review
Hsp70 and Theta Subunit of T Complex Protein , a Response to Salt Stress in the Halophyte Sesuvium Portulacastrum
- 1Department of Biotechnology, Lady Doak College, Madurai, Tamilnadu, India
- 2Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettia, Tamilnadu, India
Akshaya Murugesan, Department of Biotechnology, Lady Doak College, Madurai, Tamilnadu, India. 625 001, Tel: 91 9698913977; E-mail: firstname.lastname@example.org
Akshaya, M., et al. Hsp70 and Theta Subunit of T Complex Protein, a Response to Salt Stress in the Halophyte Sesuvium Portulacastrum. (2016) J Bioinfo Proteomics Rev 2(2): 1- 9.
© 2016 Akshaya, M. This is an Open access article distributed under the terms of Creative Commons Attribution 4.0 International License.
KeywordsHalophyte; Salt stress; Sesuvium portulacastrum; 2D electrophoresis; Mass spectrometry
The proteomic analysis was performed to explore the salt responsive proteins in S. portulacastrum a salt marsh euhalophyte. Protein profile showed profound differences in the expression pattern of salt treated plant. The total protein content was significantly high in leaf than in shoot and root. 2D electrophoresis revealed that 7 proteins were down regulated, 6 proteins were up-regulated and 46 proteins were newly expressed in S.portulacastrum leaves. Two spots from newly expressed proteins were randomly selected and characterized by MALDI-TOF/MS analysis. Protein mass fingerprinting annotation showed different peptide intensities and subsequent sequence homology search using MASCOT software categorized those proteins as Hsp70 and theta subunit of T complex protein with pI/kDa, 5.2/70kDa and 4.9/53kDa respectively. Hsp70 has been proposed as biomarker for stress produced by NaCl which emphasize its role in protecting plants against stress, whereas theta subunit of T complex protein seems to have role during protein folding expressed during abiotic stress, which yet to be characterized. The resulting findings addressed the significance of stress related proteins on the survival of plants in extreme conditions and it can be complemented with large scale studies on functional and molecular interaction of proteins expressed during saline stress.
The beginning of 21st century is marked by scarcity of drinking water resources, due to environmental pollution and increased salinization of soil and water. Almost 71% of the earth’s area is occupied by saline water. Abiotic stress is a major limiting factor in plant growth. Among the various abiotic stresses to which plants are constantly exposed, salt stress is one of the most important factor that affects growth and productivity of plants[1,2]. The plants naturally adapted to high soil salinity are known as halophytes. Their physiological process of adaptation is well developed compared to glycophytes. Salt stress adaptation in plants occurs through an organized physiological process at both cellular and molecular levels such as ion homeostasis, osmotic adjustments, ion extrusion and compartmentalization[3,4]. The Plant stress adaptive responses include dynamic transcriptome changes, presumably playing important role in co-ordination of the many different molecular events responsible for cellular homeostasis. Various genome wide analyses such as microarray, genetic and physical mapping of chromosomes, helps in identification of salt stress responsive genes. While transcriptomic approaches are important, determination of gene function requires the identification of encoded proteins by current proteomic approaches. 2D electrophoresis has been successfully applied for examination of induced gene products in several plant species subjected to a wide range of abiotic treatments, including salt, drought, temperature, ultraviolet radiation, heavy metals and herbicides. The proteomics of various plants in response to salinity have been studied[12-18], however, to date, the proteome analysis in halophytes is limited. Sesuvium portulacastrum belongs to the family Aizoaceae, commonly known as ‘shoreline purslane’. It is a sprawling perennial herb that grows in coastal areas throughout the world. It mainly inhabits sandy clay, coastal limestone, and salt marshes. It is also known for its medicinal value such as antibacterial, anticandidal and moderate antifungal activity. But the paucity of the information about the molecular regulation of salt tolerance mechanisms of S. portulacastrum persuades to choose it as a representative species for further investigation. Plant salt tolerance is controlled by complex and sophisticated molecular networks of signaling pathways. It displays common as well as specific characteristics in different species of halophytes. In the present study, S. portulacastrum was exposed to different salinities (0 mM, 100 mM, 200 mM, 300 mM, 400 mM and 500 mM NaCl) in a time course study which revealed various morphological variations and changes at protein level. Further characterization of protein of interest is done via MALDI-TOF mass spectro-photometry which explains the importance on the functions of the salt responsive protein. Our results lay a solid foundation necessary for further investigation on the salt tolerance networks.
Materials and Methods
Effect of salinity on germination of seeds
Seeds of wild type S. portulacastrum used in this study were collected from Vellar estuary (Lat. 11° 29′N and Long.79° 49′E) near Parangipettai in the southeast coast of India. Prior to germination, seeds were shade dried and hand sieved. Seeds of S. portulacastrum were immersed thoroughly in 70% (v/v) ethanol for 5 min, sterilized by 0.1% mercuric chloride and washed with sterile distilled water. Seeds were stored in 100 ml of distilled water at 27°C for 24 h. Replica of 100 seeds per 90 mm petri dishes were germinated on filter paper wetted with 3 ml of sterile ½ MS (Murashige and Skoog media, pH 5.8), and kept in culture chamber initially at 27°C for 72 h darkness followed by 16 h of photoperiod. After radical development from the seed (usually after 6 days) 3 ml of ½ MS media was added to control plate, with the increasing concentration of NaCl (100 mM, 200 mM, 300 mM, 400 mM and 500 mM NaCl) in their respective plates.
Germination (emergence of radicle) was recorded every day after start of the salt treatment. The rate of germination was calculated using GP: Ni / N × 100, where, Ni: number of germinated seed/ treatment days, N: total number of seeds. Samples were harvested after 16 days of germination and immediately stored at -80°C until use.
Plant material and saline treatment
The seeds were germinated and grown in a soil mixture containing shore sand and vermiculated manure in a 1:1 ratio in small germination plastic pots. Plants cultivated in soil were watered every day and supplemented on alternate days with a ½ MS media. Six week old plantlets were carefully transferred to hydroponic conditions and maintained in a culture room with a dark/light cycle of 8/16 h at 37°C for two weeks. Further the plants were exposed to three different concentrations of NaCl (300 mM, 0.5 M & 1 M) and subjected to time course study. Nutrient solution was replaced every day in order to avoid depletion of nutrients. Upon completion of the treatments, leaves shoot and root tissues were separately collected, frozen in liquid nitrogen at −80°C and used for various proteomic analysis.
Total Protein extraction and estimation
Total protein was extracted by phenolic method using BPP (borax/ phenol/polyvinylpyrolidine) 1 g of frozen leaf tissue from S. portulacastrum was powdered and resuspended in 3 mL of ice-cold extraction buffer containing 100 mM Tris (pH 8.0), 100 mM EDTA, 50 mM borax, 50 mM vitamin C,1% PVPP w/v, 1% Triton X-100 v/v, 2% β-mercaptoethanol v/v , 30% sucrose w/v and vortexed for 5 min at room temperature. Two volumes of tris saturated phenol (pH 8.0) were added and further vortexed for 10 min. The sample was centrifuged (Beckmann Coulter ultracentrifuge, India) at 4°C for 15 min at 15, 000 g and the upper phase was transferred to a new centrifuge tube. Equal volume of extraction buffer was added to the mixture, vortexed for 10 min, followed by centrifugation to the above mentioned condition. Proteins from the supernatant were precipitated by adding five volumes of ammonium acetate saturated-methanol, and incubated at -20°C for at least 6 h. After centrifugation as described above, the protein pellet was re-suspended and rinsed twice with ice-cold methanol followed by ice-cold acetone at 15,000 g for 5 min. at 4°C. Finally, the washed pellet was air-dried, recovered with lysis buffer (9 M urea, 2% CHAPS, 13 mM DTT, 1% IPG buffer) and stored at -80°C for further studies.
Dialysis and clean – up of protein samples: Dialysis of the protein samples was done in 50 μm thick membrane (Himedia) against dialysis buffer containing 20 mM Tris-Cl, 100 mM NaCl and 3 mM DTT (pH8.0) with change of buffer system thrice for an interval of 4 hours in cold room. After dialysis, the sample was carefully transferred to a pre-sterilized 2ml tube and further processed with clean– up kit (Bio-Rad) following the instructions given in the manual.
Quantification of Proteins: Protein concentration was determined for leaf, shoot and root of S. portulacastrum by bicinchoninic acid protein assay kit (BCA, Sigma Aldrich) following the manufacturer’s manual using the LAMBDA 25 UV/Vis Spectrophotometer (Perkin Elmer). BSA was used as the standard and the absorbance was measured at 562 nm.
Protein profiling through SDS-PAGE: Proteins were resolved in SDS–Polyacrylamide gel as described by Ausubel, F., et al. Routinely Aliquots of protein samples of equal concentration were analyzed. The precipitated proteins were recovered by centrifugation in a microfuge for 10 min. The supernatant was carefully aspirated and the pellet was dissolved in 15 μL of 0.1N NaOH and 5 μL of 4X sample buffer. The samples were heated to 80°C for 15 min, and electrophoresis was performed with a constant current of 150 volt.
Protein Separation via isoelectric focussing
One dimensional electrophoresis: An IEF-tube gel of 7 cm, with broad range pH 3.0 – 10.0) was used (Bio-Rad, USA). The strip was loaded with 100 μg of protein dissolved in 125 μL of rehydration buffer and incubated overnight for efficient rehydration. Strips are subjected to isoelectric focussing at 250 V for 20 min, 4000 V for 2 h and 4000 V/10,000 V/hr at 20°C.
Two dimensional electrophoresis: The strips were equilibrated with equilibration buffer I (50 mM Tris pH8.8, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 1% DTT, and subsequently with 4% iodoacetamide for 15 min with equilibration buffer II separation of proteins in the second dimension was performed using 10% SDS Polyacrylamide gels. The gels were stained with coomassie brilliant blue (CBB) and the positions of individual proteins on gels were evaluated robotically with PDQuest software (Bio-Rad). The pI and Mr of each protein was determined using 2D-PAGE markers.
Image acquisition and data analysis: Spot detection, spot measurement, background subtraction and spot matching were performed specifically after CBB staining of the gels using PDQuest software. Following automatic spot detection, gel images were carefully edited. Before spot matching, one of the gel images was selected as the control gel. The resulting data from image analysis were transferred to PDQuest software for querying protein spots, which show quantitative or qualitative variations. The apparent Mr of each protein in gel was determined by referencing to the protein markers.
Protein identification via mass spectrometry: Proteins were characterized according to a method described by Shen, S., et al. Briefly, individual protein spot was excised from the gel, and de-stained twice with 50% acetonitrile (ACN), 50 mM NH4HCO3 at 37°C for 30 min, dehydrated by adding 10 μL ACN, and dried. The proteins in the gel slices were reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 h, and incubated in the solution containing 40 mM iodoacetamide and 100 mM NH4HCO3 for 30 min at room temperature. Each gel slice was minced, lyophilized, and rehydrated in 100 mM NH4HCO3 containing 5 pmol trypsin (trypsin, modified, sequencing grade, Roche Applied Science, Penzberg, Germany) overnight at 37°C. Tryptic peptides were extracted two times with 20 μL 50% ACN, 0.1% formic acid (FA) for 30 min at 37°C, and 10 μL ACN for 30 min at room temperature. After each extraction, samples were centrifuged at 1000 g for 30 s, and all supernatants were combined, vacuum-dried, and stored at -80°C until MS analysis. Matrix was prepared by dissolving α-cyano-4-hydroxycinnamic acid in 50% ACN and 0.1% TFA. 10 μL of matrix solutions were added into the dried digests and the mixture was vortexed for 30 min. One micro liter was spotted initially on an Anchor chip target plate (384F, Bruker Daltonics), followed by 7 μL of matrix solution. The dried sample on the target plate was washed with 1 mL of 0.1% TFA twice, left for 30 s before solvent removal, and dried for MALDI-TOF/MS/MS analysis. Mass spectra were obtained on an Autoflex MALDI-TOF mass spectrometer (Bruker-Daltonics, Billerica, MA, USA) equipped with a pulsed N2 laser (337 nm). Then the spectrum was analyzed with flex-Analysis software (Version 3.2) and homology search was performed against non-redundant NCBI (NCBInr) database using MASCOT software Version 2.2. Best matches were selected with the Mascot score higher than 71 (threshold level). To avoid false positives, an additional in-house BLAST search at NCBI http:// blast.ncbi.nlm.nih.gov/Blast.cgiwas done to confirm all the matches.
Database searching with MS/MS spectra: MS/MS spectra were used to search against the NCBI non-redundant protein database using MS/MS Ion Search Engine, a computer software program conducting protein identification based on matching the MS/MS spectra of a protein with a protein or DNA sequence data base, MASCOT: http://www.matrixsci nce.com/help/scoring_help.html. A single protein having a higher score than the minimum score for the significance level (p < 0.05) was judged as a significant match.
In each MASCOT search output result, the minimum score for significance level was provided, based on the absolute probability and the size of the sequence database being searched. Protein sequence with the significant homologues was retrieved from NCBI database. The consensus pattern within the homologous was detected by performing multiple sequence alignment using CLC sequence viewer version 6.0. The presence of the conserved motifs was detected using conserved domain database and ScanProsite.
Results and Discussion
Effect of salinity on seed germination of S. portulacastrum: The germination percentage of halophyte was strongly affected by higher salt concentration (500 mM). At the sixteenth day after sowing, the final germination percentage for each of the different concentration of NaCl was calculated. Germination was found to be 80% in control plant after 3 days of sowing (without NaCl treatment), better at 100 mM, 200 mM NaCl and found to have significant reduction at 300 mM with the least germination percentage as the concentration of salinity increases. Increasing salinity caused delay and a gradual decrease in germination [Figure.1 (A)]. As discussed by Khajeh-Hosseini, M., et al, high salinity leads to reduced osmotic potential which prevents the water uptake necessary for the mobilization of the nutrient required for germination. It can also be due to the toxic effects of Na and Cl- ions on the germination process. Expression of protein at ~ 40kDa was found to be prominently up-regulated as the concentration of salt increases [Figure.1 (B)]. Further 2-Dimensional electrophoresis analysis was performed to quantitative the differential protein expression of seeds under salt stress.
Figure 1(A): Seeds were germinated in petri dishes on filter paper wetted with ½ MS media. The values shown are the means of germination percentage determined at different concentration of NaCl for replicates with 100 seeds per treatment.
Figure 1(B): Profiling of seed protein from S. portulacastrum by SDS-PAGE showed differential expression after treatment with varying concentration of NaCl. Lane M: protein marker; lane C: control seed maintained in ½ MS media; lane 1-4: increasing concentration of NaCl (100 mM, 200 mM, 300 mM and 400 mM) harvested after 16 days. All lanes were loaded with 20 μg protein. An arrow indicates the up-regulation of protein ~40 kDa after NaCl treatment.
Effect of salinity on plant growth: The seeds of S. portulacastrum collected from mangrove associated Vellar estuary [Figure. 2(A)] with the water salinity 26 ppt, was grown in appropriate and controlled environmental conditions in green house. During acclimatization [Figure 2 (B)], the waxy coat of the S. portulacastrum was reduced due to the sudden change in growth condition, which takes at least 4 days for its gradual recovery. Plants that are treated with NaCl above their threshold levels such as 400 mM and 500 mM for 1, 2 and 3 days, developed several stress related morphological phenotypes in leaves such as thickening of the leaves and cuticular wax depositions. Similar experiments were conducted in maize by Cramer, G.R., et al, where there is rapid and transient reduction in the rate of leaf expansion after sudden increase in salinity. This might be due to changed water relationship and not the presence of Na and Cl-.
Figure 2A: Plant acclimatized in ½ MS media in culture room for proteomic analysis.
Figure 2B: Halophyte, seeds of Sesuvium portulacastrum from the collection area were transported carefully
Effect of salinity on protein content of S. portulacastrum: Protein isolated from different tissues of S. portulacastrum such as leaves, shoot and root on varying saline treatment showed significant difference in the protein content [Figure.3]. The consistent reason is due to the abundance in the presence of Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase) in leaf tissues makes the proteins facile to be isolated by the BPP method.
Figure 3: Effect of salinity on the protein content of different tissues of S. portulacastrum at different time point. The values shown are the means of (± S.D.) of triplicate S. portulacastrum plants treated with 300 mM NaCl.
Leaf protein profiling of S. portulacastrum on NaCl treatment: Effect of salinity in the level of protein expressed in the leaves of S. portulacastrum was identified by performing two different sets of treatments with NaCl. First set of plants was treated with threshold concentrations of NaCl (300 mM) which is determined through seed germination percentage, for different time periods (4, 8, 12, 16, 20, 24, 48 and 72 h). Second set of plants was treated with higher concentration of NaCl (0.5 & 1 M) for 3, 6 and 9 h in order to study the time dependent changes in the expression of protein. Recognition of the importance of time frame led to the concept of two phase growth response to salinity. This is an important criterion when screening for the salt tolerant plants. First phase of growth reduction is apparently due to the osmotic or water stress in which there is surprisingly little genotypic variations. Second phase of growth reduction which takes time to develop which results from salt accumulating in transpiring leaves to excessive levels. This exceeds the ability of the cells to compartmentalize the salts in the vacuoles, finally leading injury to the cells. SDS-PAGE analysis was performed and the protein profile was outlined. On 10% SDS-PAGE gel, distinct protein bands were observed with varying expression levels and molecular weight. Plants treated with 300 mM NaCl showed differential expression of protein at ~54 kDa and ~15 kDa which is analyzed through alpha-imager against standard protein marker [Figure 4]. Similarly, protein extracted from the leaf of S. portulacastrum for higher concentration of NaCl showed changes in the expression of protein in abundance when compared with non- stressed control plants at 0 hour. Maximum expression was observed in 0.5 and 1 M (3 h) treatment with NaCl. As the treatment time is increased, the expression of protein at ~54 kDa & ~15 kDa [Figure 5,6] showed significant reduction, which might be due to high level of salt that makes the plant less compatible to survive in such stressful condition.
Figure 4: Protein profiling of leaf protein from Sesuvium portulacastrum 10% SDS-PAGE showed differential expression after treatment with threshold concentration of NaCl (300 mM); lane M: protein marker; lane 1-7: 300 mM salt treatment in ½ MS media at different time intervals (4hr - 72 h); lane 8 -10: direct salt treatment (300 mM Nacl) for 4h to 12 h; lane C1: control plant grown in ½ MS media; lane C2: control plant grown in vermiculated soil. All lanes were loaded with 20 μg protein. Arrows indicates the increased and decreased polypeptide expression between treated and normal plants.
Figure 5: The values shown are the means of (± S.D.) of triplicate S. portulacastrum plants treated with 0.5 M and 1 M NaCl.
Figure 6: Leaf protein profile on time dependent changes in leaves of S. portulacastrum in 10% SDS-PAGE. lane M: protein marker; lane C1: control plant grown in ½ MS media; lane C2: control plant grown in vermiculated soil; lane 1,3,5: 0.5 M treat ment at different time point (3 hr, 6 hr, 9 hr) and lane 2,4,6 : 1 M NaCl treatment at different time point (3 h,6 h,9 h).
Two-dimensional gel electrophoresis (2-DE) of S. portulacastrum leaf protein: Six week old plants were treated with 300 mM NaCl for different time periods. The Cl- content in salt treated halophytes is regulated differently roots and in leaves with minimum and maximum accumulations respectively. Since morphological and physiological differences are prominent in leaves that may play important roles in salt tolerance, we performed proteome analysis in leaves from salt-treated S.portulacastrum. Total proteins were extracted after 0 h and 24 h treatment with threshold level of NaCl (300 mM). Proteins were then separated by 2-DE, and those proteins spots showing reproducible changes were selected and their expression patterns were analyzed. 2D-PAGE analysis from proteins extracted from leaves, revealed the differential expression of 59 protein spots which showed significant difference in their abundance between control and treated samples [Figure. 7 A & B)]. Among the 59 spots, 46 were newly expressed on salt stress, 6 were up regulated and 7 were down regulated (Table 1). Most distinct two different newly expressed protein spots were selected and further functional characterization was performed through MALDI-TOF/MS.
Figure 7(A & B): 2-DE of leaf protein profile of S. portulacastrum A) Control plant grown in ½ MS media B) Plant treated with 300 mM NaCl (24 h) and proteins from leaves were extracted and separated by 2-DE. Duplicates were performed for each sample. Differentially expressed protein spots were numerically marked and the number of expressed proteins was represented in Table1. The arrows indicate the protein spots subjected for MS identification.
Table 1: Differentially expressed proteins from leaves of S. portulacastrum on treatment with 300 mM NaCl.
|Leaves of S. portulacastrum||differentially expressed proteins under salt treatment||Match between control and treated plants|
|Up regulated Down regulated newly expressed|
Of a total of 150 differentially accumulated protein spots, only 44 spots were consistent from which 2 newly expressed protein spots were selected for MALDI-TOF analysis.
Identification of salt responsive proteins from S. portulacastrum: Analysis of protein mass fingerprinting data (PMF) of two proteins (spot 1 & spot 2) derived by MS analysis using MASCOT search algorithm showed homology to heat shock protein 70 (Hsp70) from Cucumis sativus and theta subunit of T-complex protein from Coccomyxa subellipsoidea C-169. The analysis showed that the protein spot 1 [Figure. 8 (A)] functions as chaperones for newly synthesized proteins to prevent their accumulations as aggregates and folds in a proper way during their transfer to their final location. The expression of Hsp70 genes correlates positively with the acquisition of thermo-tolerance and results in enhanced tolerance to salt, water and high temperature stress in plants and for this reason Hsp70 has been proposed as potential biomarker. Prokaryotic and eukaryotic organisms respond to heat shock or other environmental stress by the induction of the synthesis of proteins collectively known as heat-shock proteins (Hsp). Hsp70 is a multifunctional protein that plays an important role in the transport of protein across membranes, in protein folding and in the assembly/ disassembly of protein complexes can direct incompetent “client” proteins towards degradation. Protein spot 2 [Figure 8 (B)] also functions as molecular chaperons that assist in folding of proteins upon ATP hydrolysis in protecting plant cell from the detrimental effects of stress. Until now, there is not much data available on the functional specificity of theta protein on salt stress. Sequence analysis for protein spot 1, hsp70 with its significant homologues from MALDI-/TOF/MS was performed. Since the S. portulacastrum genome sequences are not known, a homology based search was performed. Multiple sequence alignment with their respective homologues revealed 98% consensus pattern. Functional annotation was done using Scan prosite revealed the presence of three hsp70 protein signature pattern [Figure 9], of which penta-petptide conserved signature is present in N terminal region and the other two motifs is conserved in the central part of the sequence. Similarly, the protein spot 2 which is the theta subunit of T complex protein showed least significant among the homologues obtained via MALDI-/TOF/MS.
Figure 8(A): Identification of proteins from S. portulacastrum via MALDI TOF/MS. The protein spot 1 was excised and digested with trypsin and then collected peptide was analyzed using MALDI/TOF/ mass spectrometer. The annotated PMF spectral peaks showed the intensities of different peptide (A for spot 1). Database searching with Mascot software against NCBInr database identified the protein as Hsp70.
Figure 8(B): Identification of proteins from S. portulacastrum via MALDI TOF/MS. The protein spot 2 was excised and digested with trypsin and then collected peptide was analyzed using MALDI/TOF/ mass spectrometer. The annotated PMF spectral peaks showed the intensities of different peptide (B for spot 2). Database searching with Mascot software against NCBInr database identified the protein as T complex protein.
Figure 9: Comparison of deduced amino acid sequence with the homologues of Hsp70 obtained from Mascot software for Sesuvium portulacastrum. Sequences were aligned using the CLC sequence viewer program. Gaps have been introduced to least level to optimize the alignment. Consensus amino acids are highlighted in black boxes. All three conserved Hsp70 protein family motif are given in astrix. The sources of the proteins and gene bank accession numbers are as follows, Cucumis sativus (gi|6911551), Ricinus communis (gi|255575054), Vigna radiate (gi|45331283), Gossypium hirsutum (gi|211906496) and Arabidopsis lyrata subsp. lyrata (gi|297810345).
Plants have advantage of studying the biological responses to environmental factors as they are easily handled and also they have enormous breadth of genetic diversity at interspecific and intraspecific. Comprehensive dynamic protein profiling analysis and characterization of regulatory mechanisms are now essential to understand the plant physiology such as their adaptation and interaction with the environment. Identification of Hsp70 and theta subunit of T complex protein clearly showed the importance of such proteins during salt stress. The response of plants to stress through proteomic approach is under infancy stage and is becoming an active and evolving field with large impact on plant biology. There are various factors to be considered and addressed to develop plants salt tolerance, which may lead to application in breeding for enhanced tolerance in crop plants.
This work was supported by grants received under Centre of Excellence and programme support in areas of Biotechnology, Department of Biotechnology, Government of India, New Delhi. Thanks to the technical support by DBT, India vide project reference “BT/PR 10211/ BCE/08/615/2007”, DBT-BIF, Lady Doak College, Madurai, Tamilnadu, India.
Conflict of interest:
No Conflict of Interest.
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- 31. Yamanaka, T., Miyama, M., Tada, Y. Transcriptome profiling of mangrove plant, Bruguiera gymnorhiza and identification of salt tolerance genes by Agrobacterium functional screening. (2009) Biosci Biotechnol Biochem in press 73(2): 304-310.
- 32. Su, P.H., Li HM. Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermo tolerance of germinating seeds. (2008) Plant Physiol 146(3): 1231-1241.
- 33. Lee, J.H., Schoffl, F. An Hsp70 antisense gene affects the expression of HSP70/HSC70, the regulation of HSF, and the acquisition of thermo tolerance in transgenic Arabidopsis thaliana. (1996) Mol Gen Genet 252(1-2): 11-19.
- 34. Cho, E.K., Hong, C.B. Over expression of tobacco NtHSB70-1 contributes to drought-stress tolerance in plants. (2006) Plant Cell Rep 25(4): 349-358.
- 35. Ireland, H.E., Harding, S.J., Bonwick, G.A., et al. Evaluation of heat a. Shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna a.minor. (2004) Biomarkers 9(2): 139–155.


So, you're in love. This one's a keeper. You are…
It’s official. Your “Green Thumb” is making your neighbors “green” with envy. Your magnolias are magnificent, your tulips are tumultuous and your wisterias are without equal. So why not up the ante? While everyone is enjoying the “flowers of your labors,” the “fruits of your labors” will guarantee much sweeter returns. You’ll be saving money, saving the environment and, imagine how much better your morning grapefruit will taste when you know that it came from your own garden.
Types of Fruit Trees
Apple trees are the most popular types because they are adaptable, easy to grow, and, well, a lot of people like them. Most apple varieties, including disease resistant ‘Liberty’ and “Freedom,’ are adapted to cold hardiness, but if you are in mild inner climates, you will be better off with low chill varieties like ‘Anna’ and ‘Pink Lady. Since trees grow better next to compatible pollinators, you will want to begin with two trees to guarantee a good fruit set. Mid and late season apples generally have better flavor and store longer than early season types.
Cherries can be of a sweet or sour subset and range in color from bright yellow to nearly black. They need fertile near neutral soil and good air circulation. Twelve-foot tall cherry trees will be easy to protect from virus and birds because the smaller trees can be sprayed with sulfur or kaolin clay and can be covered by a protective net.
Citrus hybrids, like kumquat, Mandarin orange, satsuma, and ‘Meyer’ lemon are the easiest fruit to grow. The fragrant oils in citrus provide built-in pest protection, although some varieties may require extra care in the cold weather. ‘Nagami’ kumquat, ‘Owari’ Satsuma, and ‘Meyer’ lemon may need to be covered in blankets in the extreme cold, but winter citrus harvests may be worth it.
Planting Fruit Trees
Choose a sunny site with well-drained fertile soil. Dig a hole that’s two times the size of the root ball of the tree. Spread the roots in the hole, and fill with soil. Trees should be set at the same depth as they were in the nursery and care should be taken not to bury any swollen areas on the trunk. Water generously and install a trunk guard to protect the tree from rodents, scalding, and physical injuries. Stake the tree to hold steady. Mulch over the root zone with wood chips or slow rotting mulch. One year after planting, fruit tree should be fertilized. Rake back the mulch and scratch an organic fertilizer into the surface of the soil. Add a wood based mulch to a mulch depth of about 4 inches in a 4-foot circle surrounding the trees. After two years, switch from trunk guards to coating the trunks with a white latex paint, Sand will keep away voles and rabbits.
Pruning Fruit Trees
You should begin pruning fruit trees to shape them in their first year of growth and then annually in late winter. Keep in mind that pruning too much is better than too little.
Harvesting and Storage
Except for pears, fruit trees should be harvested just as they are approaching full ripeness and should be kept chilled. Apples and pears can be kept for several months in the fridge, but softer stone fruits, like nectarines, peaches, cherries and plums should be dried, frozen, canned, or juiced within the first days of being harvested.
Pests and Diseases
Apply sulfur sprays early in the season to prevent brown rot and diseases.
Are you thinking of adding a little citrus and fruit to your garden? Let us know how it’s going! We love to get your comments and suggestions.


The Finnish company founded in 1905 is the leading company in the trade of agricultural machinery in Finland, and a great reference in the European market.
Also dedicated to the supply of animal products, hardware and gardening, already has more than 64 retail outlets, 8 machinery centers, and employs about 1000 people.
Hankkija is part of the powerful Danish-Agro group, establishing itself as a strong partner for Finnish consumers and professionals.
With this agreement, Hankkija takes over the entire distribution of CORVUS UTV vehicles in Finland, and also offers users maintenance assistance and sales of spare parts and accessories.
After this agreement, Corvus takes a big step in its international growth and covers one of the European areas with more demand for agricultural and gardening machinery, and it’s glad to partner with Hankkija, ”the best possible partner for this venture.”


The Ambrosia apple
Ambrosia is a new winter Canadian apple variety, ripening in late October or early November. The fruit weighs about 215 grams, mostly red with yellow stains. The apple has a sweet taste, looks like a pear and is characterized by good winter hardiness and drought resistance, is resistant to diseases.
This variety was originated in British Columbia in the early 1990s. The first tree was revealed by the Mannell family, they found it in their garden in Similkamin-Valley, British Columbia.
he Ambrosia apple is the “club” variety, patented by the organization, which sets quality standards and provides marketing, but production is limited to club members. The name was never registered, and the patent expired in Canada and the United States.
Ambrosia is the most common in British Columbia, where it was found. It is the third-largest apple growing in the province. It is also distributed in Ontario, Nova Scotia and many other places around the world.


Jinshan Zhang, Zhenyu Zhou, Jinjuan Bai, Xiaoping Tao, Ling Wang, Hui Zhang, Jian-Kang Zhu, Disruption of MIR396e and MIR396f improves rice yield under nitrogen-deficient conditions, National Science Review, Volume 7, Issue 1, January 2020, Pages 102–112, https://doi.org/10.1093/nsr/nwz142
The microRNA miR396 directly represses GROWTH-REGULATING FACTORs (OsGRFs) and has been implicated in regulating rice yield and in nitrogen assimilation. Overexpressing the miR396 targets OsGRF4 and OsGRF6 improves rice yield via increased grain size and panicle branching, respectively. Here, we used CRISPR/Cas9 to assess the function of miR396 genes in rice. Knockout of MIR396ef (MIR396e and MIR396f), but not other isoforms, enhanced both grain size and panicle branching, resulting in increased grain yield. Importantly, under nitrogen-deficient conditions, mir396ef mutants showed an even higher relative increase in grain yield as well as elevated above-ground biomass. Furthermore, we identified OsGRF8 as a new target of miR396, in addition to the known targets OsGRF4 and OsGRF6. Disruption of the miR396-targeting site in OsGRF8 was sufficient to both enlarge grain size and elongate panicles. Our results suggest that rice-seed and panicle development are regulated by miR396ef-GRF4/6/8-GIF1/2/3 modules and that miR396ef are promising targets of genome editing for breeding environmentally friendly rice varieties that require less nitrogen fertilization.
MicroRNAs (miRNAs) are single-stranded, non-coding RNAs of 21–24 nucleotides, which typically load into a silencing complex to direct cleavage or translational inhibition, and thus repression, of complementary messenger RNAs (mRNAs) [1–3]. In plants, miRNAs act as key regulators to control development, growth and stress tolerance [4–6].
Several miRNAs have been shown to control rice yield. For example, miR156 and miR397 affect rice yield by regulating their targets, OsSPLs and OsLACs, respectively [5–9]. The microRNA miR396 was recently shown to regulate rice-grain yield by targeting the transcription factors growth-regulating factors (GRFs), which execute their function via GRF-interacting factors (GIFs) . miR396 can target and thus limit the expression of both OsGRF4 and OsGRF6 in rice . OsGRF4 controls grain development by regulating many brassinosteroid-induced genes and disruption of the miR396 target site in OsGRF4 enhances grain size and yield in rice [12,13]. OsGRF4 also balances the inhibitory activities of DELLA to promote and integrate nitrogen assimilation, carbon fixation and growth , but whether this is regulated by miR396 is not known. OsGRF6 directly promotes the expression of panicle-branching factor genes OsTAWAWA1 and OsMADS34, and decreased expression of MIR396b or overexpression of OsGRF6 increases the panicle-branching number and, in turn, grain yield . Overexpression of the miR396 target genes AtGRF1, AtGRF2 and AtGRF5 in Arabidopsis leads to the production of larger seeds, indicating that a miR396-GRF regulatory network broadly participates in seed development .
miR396 antagonizes the expression of its target GRF2 in the distal part of leaves, restricting cell proliferation in developed tissues . miR396 also negatively regulates cell proliferation by markedly decreasing the expression of cell-cycle-related genes, such as CYCLINB1;1 and TCP4 [17,18]. An miR396-GRF-GIF regulatory network has also been reported to regulate growth, development and abiotic stress tolerance in Arabidopsis [19,20]. Overexpression of miR396 in Arabidopsis reduces the accumulation of GRF7 [19,21]. GRF7 directly represses DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) and other genes that are up-regulated in response to abscisic acid; these genes enhance tolerance of dehydration and high-salinity conditions in Arabidopsis plants .
Given that a single miRNA often targets multiple genes to regulate diverse biological processes, identifying and manipulating key miRNAs is considered a potential strategy to improve agronomic traits of crop plants . Gene-editing technology has been applied to improve agronomic traits and we were interested in using this approach to manipulate key miRNAs and generate resources for breeding elite crop varieties. Here, we studied the individual functions of the eight members of the miR396 gene family in rice. We discovered that two members, miR396e and miR396f (miR396ef), regulate seed and panicle development. mir396ef mutants showed increases in grain size and panicle branching, which resulted in increased grain yield. Importantly, mir396ef mutations showed a more substantial increase in grain yield and above-ground biomass under conditions of nitrogen deficiency. We demonstrated that OsGRF4, OsGRF6 and OsGRF8 are the main targets of miR396ef and that miR396-GRF4/6/8-GIF1/2/3 modules regulate rice-grain and panicle development. Our work suggests that miR396ef is an attractive target for generating high-yield rice plants that are less dependent on nitrogen fertilization.
miR396ef isoforms regulate grain development in rice
Rice contains five different miR396 isoforms, encoded by eight genes (Fig. 1A). According to the plant miRNA database PmiREN (Plant miRNA ENcyclopedia, http://www.pmiren.com/), the family members MIR396e and f have the highest expression in reproductive tissues, such as inflorescence, florets and spikelets (Supplementary Table 1) [23,24]. MIR396c is also expressed in these reproductive tissues, but at a relatively lower level (Supplementary Table 1) [23,24]. To identify the miR396 isoforms that modulate rice-seed and panicle development, we used CRISPR-Cas9 and five sgRNA constructs to knock out the eight MIR396 members (Supplementary Fig. 1). miR396a–c and miR396e–f have the same mature sequences, respectively, and each group was targeted by one construct. Although miR396 d, g and h also have the same mature sequence, they are located on the complementary strands of different genes, and therefore were targeted by three different constructs to avoid functional disruptions of overlapping genes (Supplementary Fig. 1D).
CRISPR lines with mutations in mature miR396 regions, which should disrupt function, were selected for further study. Unexpectedly, only mir396ef mutants exhibited grain-size phenotypes, whereas the remaining mir396 mutants displayed no differences compared with wild-type (WT) (Fig. 1B and Supplementary Figs 2 and 3). In addition, mir396abc triple mutants, mir396d and mir396g single mutants (with non-frameshift mutations in overlapping genes) and mir396h mutants exhibited no differences in morphological phenotypes compared to WT, including plant height (Supplementary Figs 2A and 3A, E and I), grain length, grain width (Fig. 1B and Supplementary Figs 2B and C and 3B, C, F, G, J and K) and panicle architecture (Supplementary Figs 2D and E and 3M and N). We also applied the short tandem target MIMICs (STTM) technology to block miR396ab functions (Supplementary Fig. 4A and E). The STTM-miR396 lines showed similar seed length, seed width and panicle branching compared with WT (Supplementary Fig. 4B–D).
In contrast, compared with the WT, mir396ef mutants showed obvious increases in grain length, width and thickness (increased 8.46%, 7.95% and 8.16%, respectively) (Fig. 1B and C). The 1000-grain weight was also significantly increased in mir396ef (31.78 ± 0.55 g) compared to WT (25.12 ± 0.30 g) (Fig. 1D). We obtained many mir396ef double-mutant CRISPR lines with different genotypes, all of which displayed the same seed phenotypes, confirming the functions of MIR396ef during seed development (Fig. 1B and Supplementary Fig. 5A and B). We generated mir396abcef mutants by crossing mir396abc with mir396ef mutants, which showed similar grain length and width as mir396ef (Fig. 1B). The mature isoform of miR396ef was almost undetectable in mir396ef leaf and flower (Fig. 1E and F), indicating successful elimination of MIR396e and f.
Due to the extremely high genome-editing efficiency at the mi396e and f target sites, we obtained only mir396ef double mutants. To obtain an mir396e single mutant, we designed an sgRNA to specifically disrupt MIR396e (Supplementary Fig. 6A and B). The grain length and width of mir396e mutants are 4.77% and 5.58% larger than the WT, respectively (Supplementary Fig. 6C and D). The seed phenotypes of mir396e are weaker than that of mir396ef double mutants (Supplementary Fig. 6C), suggesting that both MIR396e and MIR396f contribute to the grain-size development. We examined putative off-target loci for miR396abc and miR396ef target sequences and did not observe any mutations (Supplementary Fig. 7). These results indicate that both miR396e and f isoforms are involved in rice-grain development, although mir396f single mutants are needed to confirm the function of miR396f.
mir396ef mutants display simultaneous increases in grain size and panicle branching
The increased seed size of the mir396ef mutants was associated with a significantly enlarged grain husk, as demonstrated by both histological sectioning and scanning electron microscopy (Fig. 2O and P). The length and number of inner palea and lemma cells were significantly increased in the mir396ef grain husk compared with the WT (Fig. 2M and N). An MIR396e-promoter::GUS assay indicated that MIR396e was expressed in grain hull, spikelet, leaf and root (Supplementary Fig. 8), consistently with its regulation of grain-husk development.
We found that mir396ef mutant plants (74.13 ±3.54 cm) were taller than the WT (69.33 ± 3.20 cm), though the tiller number was similar to WT (Fig. 2A–C). In addition, compared to the WT flag leaf, the mir396ef flag leaf was 32.8% longer but had the same width (Fig. 2D, G and H), resulting in an increased leaf area. The photosynthetic rates of the flag leaf showed no difference between the mir396ef and the WT in the paddy field (Supplementary Fig. 9). The unchanged photosynthetic rates with increased leaf area would enhance overall photosynthesis, perhaps leading to the seed increase in mir396ef mutants relative to WT.
Rice grains grow on the panicle with numerous branches. Longer panicles and more panicle branches generally correlate with more grains. Compared to WT, mir396ef lines had longer panicles (20.55 ± 1.83 vs 17.93 ± 1.64 cm), more primary branches (8.8 ± 1.34 vs 7.33 ± 1.56) and higher grain numbers per panicle (77.82 ± 8.89 vs 72.35 ± 10.96) (Fig. 2E, F, I, J and K). However, the mir396ef panicles were partly enwrapped by the flag leaf, which affects the fertility and filling of the enwrapped panicle (Supplementary Fig. 10A–C).
The phenotypes of mir396ef, including a longer flag leaf for enhanced photosynthesis, larger grain size for more grain weight and more grains per panicle, suggest that mir396ef could be valuable targets to increase grain yield. Our paddy-field plot-yield test revealed an approximately 4% increase in grain yield on conventional agricultural 270 kg ha−1 nitrogen fertilizer (Fig. 2L). Like WT mature pollens, mir396ef pollens were deeply stained by iodine–potassium iodide (I2-KI) and their fertility was not changed (Supplementary Fig. 10D–F). The yield increase of 396ef was below what we expected, perhaps due to a higher rate of empty grains in the mir396ef mutants than in WT (Supplementary Fig. 10C).
We also tested grain-quality traits in the mir396ef mutants. The brown and polished grains of mir396ef mutants had a more ‘white splotch’ appearance compared with the WT-grain appearance (Supplementary Fig. 11A and B). The alkali-spreading value (gelatinization temperature) as well as the percentage and degree of chalkiness were increased (Supplementary Fig. 11C, D and F), whereas the gel consistency was decreased in the mir396ef mutants compared with the WT (Supplementary Fig. 11E).
mir396ef mutants exhibit significantly increased grain yield under nitrogen-deficient conditions
The miR396 target OsGRF4 confers increased nitrogen assimilation and biomass accumulation in rice . To investigate the response of miR396 to nitrogen treatments, we examined the expression of miR396 family members in seedlings grown in hydroponic culture either with or without nitrogen supply. We found that the levels of miR396f increased transiently in seedlings grown under nitrogen-deficient conditions, reaching a maximum at about 6 hours after exposure to nitrogen deficiency, then decreasing and returning to normal levels at about 24 hours (Supplementary Fig. 12). In contrast, miR396e expression did not change in response to nitrogen deficiency (Supplementary Fig. 12).
Next, we examined the nitrogen assimilation of the mir396ef mutants at the rice-seedling stage. We found that seedlings from mir396ef plants had a significantly higher nitrogen content than those from WT plants, when grown in hydroponic culture either with or without nitrogen supply (Fig. 3A and B). Recently, Li et al. showed that OsGRF4 recognizes the GCGG core motif by ChIP-Seq; ChIP-PCR and Electrophoretic Mobility Shift Assay (EMSA) confirmed that OsGRF4 associates with promoters that contain this motif and regulate genes involved in ammonia and nitrate metabolism, including GOGAT2, NIR1 and GS1.2, etc. . To determine whether genes involved in nitrogen assimilation and utilization are differentially expressed in mir396ef mutant and WT seedlings, we performed reverse-transcription quantitative PCR (RT-qPCR). Specifically, we examined the expression of NIR1, NIR2, GOGAT2 and GS1.2, which encode enzymes involved in nitrogen assimilation . We also examined the AMINO ACID PERMEASE genes (OsAAPs), which are nitrogen-use-efficiency-related genes that determine amino-acid delivery from source to sink in plants [25,26]. All of these genes we examined showed increased expression in mir396ef mutant seedlings compared to WT seedlings (Fig. 3C and D). These results suggest that MIR396ef is involved in nitrogen assimilation and utilization in rice.
Next, we determined whether the improved nitrogen assimilation and utilization of mir396ef mutant plants affect growth and yield under nitrogen-deficient conditions. We found that mir396ef mutants grew substantially taller than WT plants when grown in hydroponic culture without nitrogen supply (Fig. 3A). Moreover, mir396ef plants showed a substantial yield increase (∼15%) compared with the WT under nitrogen-deficient conditions (0 kg ha−1 nitrogen fertilizer application) in the paddy field (Fig. 3E and F). The yield of mir396ef under low-nitrogen conditions was comparable to the yield of WT under normal nitrogen-fertilization conditions (Supplementary Table 2). Interestingly, mir396ef showed a 25% increase in above-ground dry biomass per plant under nitrogen-deficient conditions compared to WT plants (Fig. 3G).
Regulation of grain size and branch length by an miR396ef-GRFs-GIFs module
Twelve GRF transcription factors in rice harbor the miR396 target site . We found that only OsGRF4, OsGRF6 and OsGRF8 were up-regulated in mir396ef plants compared to WT (Fig. 4A). RLM-RACE (5′ RNA ligase mediated rapid amplification of cDNA ends) analysis showed that miR396 could direct the cleavage of OsGRF4 and OsGRF6 mRNAs in vivo at a specific site within the miR396 pairing region with leaf samples (Supplementary Fig. 13A). Interestingly, in leaf samples, miR396 caused the cleavage of OsGRF8 mRNA at a site 118 bp downstream of the miR396 pairing region, and results of transient co-expression assays in Nicotiana benthamiana leaves are consistent with such a cleavage (Supplementary Figs 13A and 14). However, in flower samples, miR396 could direct the cleavage of OsGRF8 mRNA at the predicted miR396 pairing region (Supplementary Fig. 13A).
To investigate whether miR396-mediated regulation of OsGRF4, OsGRF6 and OsGRF8 control seed and panicle development, we constructed miR396 target-resistant versions of OsGRF4, OsGRF6 and OsGRF8 (designated np:rGRF4, np:rGRF6 and np:rGRF8, respectively) (Fig. 4B) and introduced them into rice under the control of their respective native promoters. We found that np:rGRF4, np:rGRF6 and np:rGRF8 lines exhibited larger grain sizes than WT plants (Fig. 4C and Supplementary Fig. 13B–D), with np:rGRF4 showing the largest increase and np:rGRF6 showing the smallest increase (Fig. 4D and Supplementary Fig. 13B–D). Both np:rGRF6 and np:rGRF8 lines showed increased panicle lengths (Fig. 4E and F and Supplementary Fig. 13I), but their branching numbers remained unchanged compared to WT. The np:rGRF4 lines displayed similar panicle length and branching numbers to WT (Supplementary Fig. 13H). These results suggest that the miR396ef members regulate seed and panicle development by regulating their target genes OsGRF4, OsGRF6 and OsGRF8.
GRFs have been shown to interact with transcription coactivators GIFs [12,20]. To uncover GIFs that interact with OsGRF4 and OsGRF6, we performed a yeast two-hybrid assay. We found that OsGIF1, OsGIF2 and OsGIF3 interacted with OsGRF4, and OsGIF3 also interacted with OsGRF6 (Fig. 5A). Biomolecular fluorescence complementation (BiFC) assays confirmed these interactions in tobacco leaves (Supplementary Fig. 15). The expression of OsGIF1 was significantly increased in mir396ef plants (Supplementary Fig. 16A). We used the CRISPR/Cas9 technology to generate gif1 mutants and observed the expected short-leaf phenotype (Supplementary Fig. 16B). osgif1 mutants also exhibited smaller plant stature and aborted seed phenotypes compared to WT (Supplementary Fig. 16B and C). These findings suggest that rice-seed and panicle development are regulated by the miR396ef-GRF4/6/8-GIF1/2/3 module.
To further explore the function of miR396ef in plants, we used RNA-sequencing analysis to examine the transcriptomes of leaves and young spikelet tissues from mir396ef and WT plants. We detected 558 DEGs (differentially expressed genes, ratio > 1.5) in young spikelet tissues, but only 189 DEGs in leaves. We observed 314 and 127 up-regulated genes in mir396ef mutant spikelet and leaves, respectively, compared to WT. In addition, 244 and 62 genes were down-regulated in mir396ef mutant spikelet and leaves compared to WT, respectively (Supplementary Fig. 17A). We observed 15 DEGs encoding ‘nitrogen-related genes’ in leaves and enrichment analysis of the DEGs identified ‘nitrate ion transport and metabolism’ as the top annotated category (Fig. 5B and C). In addition, 26 DEGs were related to brassinonsteroid-induced genes and 21 DEGs were related to auxin signaling, consistently with auxin- and brassinosteroid-mediated regulation of spikelet number and grain size (Supplementary Fig. 18A and B) [12,15]. We confirmed the expression of key genes within brassinosteroid and auxin-signaling pathways by RT-qPCR (Supplementary Fig. 19A and B). We also observed increased expression of the cell-proliferation-related genes, two CYCLINB1;1 homologous genes and one TCP4 homologous gene, in mir396ef mutant, consistently with the increases in cell number (Supplementary Fig. 19C–E).
Here, we investigated the role of the miR396 gene family in controlling grain yield in rice, with the goal of identifying targets and strategies for breeding elite crop varieties. The miR396 target gene OsGRF4 increases grain size by activating brassinosteroid responses , whereas the miR396 target gene OsGRF6 promotes panicle branching through the activation of several auxin biosynthesis factors . Thus, targeting miR396 itself would be expected to enhance both grain size and panicle branching. We examined the miR396 members by CRISPR/Cas9 editing (Fig. 1A and Supplementary Fig. 1) and found that mir396ef mutants showed the expected traits of increased grain size and panicle branching. It was reported that miR396b negatively regulates panicle branching by targeting OsGRF6, and STTM-miR396b plants showed increased panicle branching and grain yield . However, in our data, STTM-miR396ab lines showed unchanged panicle branching compared with the WT. This discrepancy may be caused by the different genetic backgrounds, since the reported STTM-miR396b lines were in the indica background whereas our STTM-miR396ab lines are in the japonica background. More recently, Miao et al.  reported that mir396e single mutant rice had a similar grain size to the WT Xiushui 134. However, in our data, the grain length and width of mir396e mutants are larger than the WT Nipponbare, which may also be explained by the background difference.
We found that mir396ef mutants have several beneficial traits relative to WT plants, including increased plant height (Fig. 2A and B), enhanced length of the flag leaves and panicles (Fig. 2D and E), increased primary branch and grain number in panicle (Fig. 2F, J and K) and increased grain size and weight (Fig. 1B–D). Furthermore, the cell length and cell number in the grain hull of mir396ef mutants are both increased, which enlarged the sink size. Just as in OsGRF4-overexpression grains, the mir396ef mutant showed decreased grain quality because of increased chalkiness (Supplementary Fig. 11), which could be offset by alleles of high-quality genes .
miR396 attenuates cell proliferation in developing leaves and negatively regulates leaf growth in Arabidopsis thaliana [17,27]. We found that mir396ef mutant rice plants also showed an obvious increase in leaf length. In addition, the np:rGRF4, np:rGRF6 and np:rGRF8 lines all exhibited increased leaf length and enwrapped panicles (Supplementary Fig. 13E–G). The np:rGRF4 plants are obviously shorter than the WT, perhaps because the np:rGRF4 panicles were severely enwrapped by the flag leaf (Supplementary Fig. 13E). The significant increases in the source leaves of mir396ef mutants could explain the increased grain yield and biomass. Although the grain yield of mir396ef mutants was only 4.0% greater than that of WT in test plots with normal nitrogen fertilization (Fig. 2L), it was 15.0% greater in natural paddy fields without nitrogen fertilization (Fig. 3E and F). Further, the above-ground biomass of mir396ef mutant plants was 25.0% more than that of WT plants under nitrogen-deficient conditions (Fig. 3G). These results suggest that mir396ef mutations cause enhanced nitrogen utilization, consistently with increased OsGRF4 function, which has been shown to balance the inhibitory activities of DELLA to promote and integrate nitrogen assimilation, carbon fixation and growth (Fig. 3C and D) . This notion is supported by our discovery that nitrogen transport and metabolism genes are up-regulated in mir396ef mutants (Fig. 5B and C).
OsGRF4 and OsGRF6 have been reported as targets of miR396ef. Based on expression and mRNA cleavage studies, we uncovered OsGRF4, OsGRF6 and OsGRF8 as major targets of miR396ef (Fig. 4A and Supplementary 13A). Plants expressing miR396ef-resistant versions of target genes, np:rGRF4, np:rGRF6 and np:rGRF8, all exhibited larger grain size to varying degrees, with the largest increase in np:rGRF4. However, the panicle length and branching numbers of np:rGRF4 were unchanged compared to WT (Fig. 4C and D and Supplementary Fig. 13E). Panicle lengths were increased in np:rGRF6 and np:rGRF8 (Fig. 4E and F), but branching numbers were unaffected (Supplementary Fig. 13H and I). In summary, we did not observe synchronous increases in grain size and panicle branching in np:rGRF4, np:rGRF6 and np:rGRF8 plants. These data suggest that the seed-size and panicle-length improvement of mir396ef mutants is due to the combined functions of OsGRF4, OsGRF6 and OsGRF8.
We found that GIFs OsGIF1, OsGIF2 and OsGIF3 interact with OsGRF4, and OsGIF3 also interacts with OsGRF6 (Fig. 5A). Knockout of OsGIF1 led to an expected short-leaf phenotype, suggesting that miR396ef-GRFs-GIFs modules directly control the development of seeds, panicles and leaves in rice. Previous studies demonstrated that miR396 regulates seed and panicle development through brassinosteroid and auxin-signaling pathways [12,15] and our RNA-seq data revealed an altered expression of many brassinosteroid-induced genes and auxin-signaling genes in the mir396ef mutant (Supplementary Fig. 18). We also found that np:rGRF4 and np:rGRF6 have enlarged leaf angles, exhibited as losing plant structure, which are typical phenotypes of brassinosteroid pathway mutants, such as m107 and Gi-2 . These data suggest that miR396ef might influence rice architecture by activating brassinosteroid responses.
Taken together, our findings reveal that manipulation of miR396ef can simultaneously improve grain size and panicle branching. miR396ef thus represents a promising target to increase grain size and grain yield, especially in nitrogen-deficient environments, which might help breeders develop environmentally friendly elite rice varieties.
Materials and methods
Plant materials and growth conditions
The rice variety Nipponbare (Oryza. Sativa L. spp. Japonica, var Nipponbare) was used in this study. Rice plants were cultivated under field conditions at two different experimental stations located in Shanghai (30°N, 121°E) and Lingshui (Hainan Province, 18°N, 110°E). The phytotron, with a 30/24 ± 1°C day/night temperature, 50%–70% relative humidity and a light/dark period of 14 hours/10 hours was used to culture rice seedlings.
Nitrogen-deficiency response assay
Nitrogen deficiency in hydroponic culture conditions was modified from previous work . Seeds were disinfected in 20% sodium hypochlorite solution for 20 min, thoroughly washed with deionized water and then germinated in a dish with sterilized water. The germinated grains were then selected and transplanted to PVC culture pots that contained hydroponic nutrient solution (0.5 mM NaH2PO4, 0.75 mM K2SO4, 1 mM CaCl2, 1.667 mM MgSO4, 40 μM Fe-EDTA (Na), 19 μM H3BO3, 9.1 μM MnSO4, 0.15 μM ZnSO4, 0.16 μM CuSO4 and 0.52 μM (NH4)3Mo7O24, pH 5.5) with or without 1.25 mM NH4NO3. All nutrient solutions were changed every 2 days. The temperature was maintained at 30°C day and 24°C night, and the relative humidity was 70% in phytotron.
The CRISPR-Cas9 vectors targeting MIR396abc, MIR396d, MIR396ef, MIR396g, MIR396h, MIR396e and OsGIF1 were constructed as previously described  and the oligos used are listed in Supplementary Table 3. To construct the miR396-resisitant vectors for OsGRF4, OsGRF6 and OsGRF8, the genomic sequences of OsGRF4, OsGRF6 and OsGRF8, fused with the OsGRF4 promoter, OsGRF6 promoter and OsGRF8 promoter, respectively, were amplified and the synonymous mutations were introduced in the miR396 target sequences. All the vectors described above were used to transform rice Nipponbare by Agrobacterium tumefaciens-mediated methods.
Phenotyping and histological experiments
Plant materials were photographed using a Canon EOS7D digital camera and observed using an OLYMPUS BX53 microscope. Grain size was analysed using an SC-A grain analysis system (Wseen Company, China).
Young rice grains were collected and fixed overnight at 4°C in FAA (50% ethanol, 10% formalin and 5% acetic acid) solution and dehydrated in a graded ethanol series. The samples were then embedded in Technovit 7100 resin (Hereaus Kulzer) and made into 2-μm sections using a Leica RM 2265 programmable rotary microtome (Leica Microsystems). After being stained with 0.05% Toluidine Blue, transverse sections were photographed using an OLYMPUS BX53 microscope. Dehydrated grain-husk samples were stocked on the copper stub and sputter-coated with gold-palladium. The seed samples were examined under a scanning electron microscope, JSM-6360LV. Cell number and cell length in the outer parenchyma layer of the spikelet hulls were measured using the OLYMPUS stream software.
Total RNA was isolated using the Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. About 40 μg of total RNA from leaves and flowers were analysed on a denaturing 19% polyacrylamide gel, transferred to Nytran Super Charge Nylon Membranes (Schleicher & Schuell BioScience) and cross-linked using a Stratagene UV Crosslinker. DNA oligonucleotides complementary to different sequences of miRNAs were synthesized and labeled with [32P]-γ-ATP (PerkinElmer) using T4 polynucleotide kinase (TaKaRa). The membranes were pre-hybridized with PerfectHyb (Sigma) hybridization solution and then hybridized with the labeled probes. After several times of washing, the membranes were autoradiographed using an X-ray film (Carestream, X-OMAT BT Film). For RNA loading, 18S and 5S rRNAs were used as controls. The probe sequences are listed in Supplementary Table 3.
Plants of the WT and mir396ef were grown in Shanghai paddy fields under natural conditions. The area per plot was 2.0 × 2.0 m and 100 plants were cultivated in each plot with planting density of 20 × 20 cm. The nitrogen fertilizer was 270 kg ha−1 in the control field, whereas the nitrogen-deficient fields were without nitrogen fertilizer. Plants were irrigated with river water.
RNA extraction and RT-qPCR and stem-loop PCR
Total RNA was isolated from roots, leaves and flowers from different developmental stages of rice plants. The RNA extraction followed the method mentioned above. After being treated with RNase-free DNase I (Promega), total RNA (1 μg) was reverse transcribed using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen Biotech). The reverse-transcription products were used as templates for RT-qPCR performed on a CFX96 real-time PCR system (Bio-Rad) using SYBR Premix Taq (TransGen Biotech) according to the manufacturer’s protocol. ACTIN1 was used to normalize gene expression and OsU6 was used in microRNA expression. Relative expression levels were measured using the 2–ΔΔCt analysis method. The primers used in RT-qPCR or stem-loop RT-qPCR are listed in Supplementary Table 3.
Transient expression assays in N. benthamiana leaves
The CDS of OSGIF1, OsGIF2 and OsGIF3 were fused in frame with nYFP (yellow fluorescent protein) sequence. The CDS of OsGRF4, OsGRF6 and OsGRF8 were fused in frame with cYFP sequence. nYFP-OsGIF1, nYFP-OsGIF2, nYFP-OsGIF3, cYFP-OsGRF4, cYFP-OsGRF6 and cYFP-OsGRF8 constructs were transformed into Agrobacteria. These stains containing nYFP and cYFP plasmids were co-infiltrated into leaves of N. benthamiana. YFP signals were detected by confocal microscopy LSM800 (ZEISS). The full-length and C-terminal deletion cDNAs of OsGRF8 were fused in frame with eGFP (enhancer Green Fluorescent Protein) sequence and driven by the 35S promoter. The stem-loop sequences of miR396e and miR396f were cloned and inserted after the 35S promoter. nYFP-OsGIF1, nYFP-OsGIF2, nYFP-OsGIF3, cYFP-OsGRF4, cYFP-OsGRF6, cYFP-OsGRF8, 35S::GRF8-eGFP, 35S::GRF8(1–574)-eGFP, 35S::GRF8(1–694)-eGFP, 35S::miR396e and 35S::miR396f constructs were transformed into Agrobacteria. These stains were co-infiltrated into leaves of N. benthamiana. YFP and GFP signals were detected by confocal microscopy LSM800 (ZEISS).
Yeast two-hybrid assay
The prey vector pGADT7 and bait vector pGBKT7 were used in the yeast two-hybrid assay. Full-length cDNAs of OSGIF1, OsGIF2, OsGIF3, OsGRF4, OsGRF6 and OsGRF8 were amplified and separately cloned into pGADT7 and pGBKT7. The prey and bait plasmids were co-transformed into the yeast AH109 strain and grew on SD-Leu-Trp solid media (Clontech). The strains were transferred to the SD-Ade-His-Leu-Trp solid media to test the interactions between prey and bait.
RLM-RACE was performed with the FirstChoice™ RLM-RACE Kit (Ambion). In general, total RNA was extracted from samples and the first and second PCRs were performed, with the primers of OsGRF4/6/8-inner and OsGRF4/6/8-outer (Supplementary Table 3), respectively. The products from the second PCR were purified by agarose gel electrophoresis and then cloned for sequencing. For OsGRF4 and OsGRF6, leaf samples were used in these experiments. For OsGRF8, flower samples were also used in these experiments.
Total RNA was extracted using the RNeasy Plant mini Kit (Qiagen) according to the manufacturer’s instructions. Two independent replicates were used for each sample. The cDNA synthesis, purification and labeling were then performed following the standard protocols. The RNA-seq data were analysed with TopHat and Cufflinks software. Genes with at least 1.5-fold up- and down-regulation in the mir396ef plants compared with those in WT plants were considered as DEGs.
Net photosynthetic rate measurements
The net photosynthetic rate was measured using an LI-6400XT (LICOR Biosciences) at a light intensity of 1000 μmol m−2 s−1 and the reference CO2 concentration was 400 μmol mol−1. The measurement followed the method from the LI-6400XT manual. Two leaves were used in one measurement and placed side by side in order to fill the area of the measure room because the rice leaves were narrower than the diameter of the measure room. The net photosynthetic rate was calculated and recorded by the machine. All experiments were conducted with at least five replicates.
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27040101 to J.-K.Z.), Postdoctoral Science Foundation Grant of China (2017 M621553 to J.Z.), National Natural Science Foundation of China (31801016 to J.Z. and 31871223 to H.Z.) and Shanghai Pujiang Program (18PJ1411000 to H.Z.).
J.Z., Z.Z., J.B., X.T. and L.W. performed the experiments and analysed the data. J.-K.Z. and H.Z. supervised the project, designed the research and participated in data analysis. J.Z., H.Z. and J.-K.Z. wrote the paper.
Conflict of interest statement. None declared.
Equally contributed to this work.


Gorgie City Farm, Edinburgh
Love Farm A nimals then visit Gorgie City Farm
At Gorgie City Farm, we provide a showcase of traditional farming methods from Scotland and beyond, giving you the chance to get hands-on, practical knowledge of how to keep farm animals happy and how to care for them properly. Although our animals move around throughout the year, you will often find pigs, cows, chickens, sheep and goats on the farm.
- Street Address: 51Gorgie Road
- City: Edinburgh
- County: Edinburgh


- This event has passed.
Are You Hungry For More?
August 16 @ 10:00 AM
An event every week that begins at 10:00am on Sunday, Friday and Saturday, repeating until August 31, 2019
Are You Hungry for More?
Fridays, Saturdays, and Sundays, August 2 – 31,
drop-in 10am – 1pm
There are more than tomatoes and beans to eat. Come join our tour and
discover the myriad of both cultivated foods and edible wilds. Discover what
it takes to get food to our table and explore the new Edible Garden.
You’re sure to leave hungry for more!


Agroforestry is the combination of crops (plants, animals, fungi) and trees in forest-inspired agricultural systems that benefit both humans and nature. However, without proper design examples it is hard to sustain. Luckily, Backyard Abundance, an environmental education non-profit organization, has now released an open-source document on how to create your own “food forest” through what is called, Edible Agroforestry.
Human communities benefit through a greater connection to landscapes, improved stewardship of resources, and enhanced economic opportunities. Not to mention it is a low-maintenance plant-based food production which replicates natural ecosystems and could possibly even supply us with free food one day.
Currently, growers are confronted with a huge barrier of time, money, and risk associated with researching planting configurations, estimating implementation costs and payback, and experimenting with planting techniques without proper design examples of best-practices.
The “Edible Agroforestry Designs” document provides design templates of edible agroforestry practices to aid growers with the design, implementation, and management of environmentally-beneficial ecosystems that support personal income and community needs.
These so-called food forests are so popular that even New York has its own mobile floating food forest.
H/T to themindunleashed
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Posted by : Ivy Hamilton dimanche 23 mars 2014
Weevil is the common name for certain beetles of the snout beetle family (Curculionidae) -- small, usually dull-colored, hard-bodied insects, very common.The mouthparts of snout beetles are modified into down-curved snouts, or beaks, adapted for boring into plants; the jaws are at the end of the snout. I always think they look like a mini elephant myself. The bent antennae usually project from the middle of the snout. The largest weevils are about 3 in. (7.6 cm) long, with the average length being about 1/4 in. (0.6 cm). This one was about 4mm in length so you're looking at between half and 1 mm worth of weevil. Weevils come in an assortment of bright colors but are remarkably small and hard to see. The snout varies greatly in length among the different species; in the curculios, or nut weevils, it may be longer than the body.
Many weevils are damaging to crops. The grain or wheat weevil (Sitophilus granarius) damages stored grain. Weevils can be found in dry foods including nuts and seeds, cereal and grain products, such as pancake mix. In the domestic setting, they are most likely to be observed when a bag of flour is opened. Their presence is often indicated by the granules of the infested item sticking together in strings, as if caught in a cobweb. But, all in all, weevils are a rare occurrence in the British home. They can be removed and the area sanitised with ease. Weevils in the garden, however, can cause serious damage, especially in the early summer months when grubs are hungry and seeking food. They can become a problem if left untreated. Weevils are harmless to humans. In fact the weevil family has more species in it than any other group of organisms: about 50,000 species. You can find weevils almost everywhere but especially by looking underneath leaves in the springtime evening or by noticing signs of damage to plants. Or, as was the case with us, old birdseed at the bottom of a container in the piggery outside (shed), it was crawling with these.


We’ve planted a garden! Suzanne, our Guest Services Manager, is an avid gardener with a very green thumb. She grew tomatoes in her own garden last year, and decided that our romantic Los Angeles bed and breakfast hotel could use a dose of homegrown vegetables for our homemade fare.
So far our herb garden, growing in two planters located on our courtyard patio, consists of spearmint, Italian oregano, Italian basil and parsley. We use the herbs in our soufflés, as flavor for our homemade dips, and as garnishes for our wine and cheese spread and fresh fruit platters. We’ve also used the mint to make hot mint tea and have tossed a few sprigs in our lemonade. Ask us to make you a cup!
In our rose garden (behind the white picket fence in our parking lot), Suzanne has filled two Earthboxes with all organic dirt mix and fertilizer. One box contains a Black Cherry Heirloom Tomato plant and a Yellow Brandywine Heirloom Tomato plant. The second Earthbox has two zucchini plants and one cucumber “Burpless Diva” plant. Once we have some fresh veggies, you can expect to enjoy them at wine and cheese (in dips, as well as on the vegetable platter) and on our savory breakfast soufflés.
Suzanne chose Earthboxes for our tomato and zucchini plants because the design of the boxes makes them very water efficient. A cover is placed over the top soil to prevent excess evaporation and the water is held in a reservoir at the bottom of the planter so none of the water is wasted in run off. How very “green” of us!
|Our homegrown zucchini plants soaking up the sun!|
Besides the upkeep of our beautiful plants, our challenge is to keep the California Ground Squirrels from dining on the proceeds from our garden every night. You can see them roaming around in the Wetlands, and these little critters like to eat!
Below is a delicious recipe you can try at home:
Tomato Feta Dip
2-3 Tbsp Olive Oil
20 Grape Tomatoes – cut in half
Salt, pepper, & salt-free herb seasoning (to taste)
½ c. Feta Cheese (approx.)
Heat a small frying pan and add olive oil. Add tomatoes, salt, pepper and seasoning and cook until soft. Crush with a wooden spoon until it makes a chunky sauce. Remove from heat and cool slightly. Add oregano and Feta cheese and combine gently. Serve warm.
Do you have a favorite recipe to share with us? Please email firstname.lastname@example.org. We’ll give it a try! Bon Appétit!


Hop harvest is now upon us. Farmers in the Yakima Valley are just now beginning to harvest the crop in earnest, but a couple weeks ago Joel Vandenbrink, owner/brewmaster at Two Beers Brewing, drove a box truck to Mabton, Washington where he procured some of the very first hops of the season.
After immediately returning the bounty to Seattle, a crew of volunteers assembled on the loading dock at the brewery to strip the cones from the bines. soon thereafter, the hops realized their ultimate destiny and Washington’s first fresh hop beer of 2015 was brewed. (See the photos below, by Mark B. Bauschke Photography.)
On Friday, August 21, Two Beers Brewing releases the first fresh hop beer of the season, the 2015 version of its Fresh Hop IPA, at The Wood Tasting Room in Seattle’s SoDo district.
[singlepic id=1155 w=440 h= float=none]
“Our fresh hop beer is a true labor of love and we’re proud to release the first fresh hop of the season,” said Joel VandenBrink, founder of Two Beers Brewing Co. “This year, we had more vines than ever before and more than 40 volunteers on the dock ready to help us pick the hops. It’s rare to be able to have your fans be the ones who help create a beer, which is one of the main reasons Fresh Hop is our favorite beer of the season.”
[singlepic id=1151 w=440 h= float=none]
Ringing in at approximately 80 IBUs and 6.2 percent ABV, the 2015 Fresh Hop IPA’s strong citrus aroma – offering hints of grapefruit and passion fruit – can be attributed to the freshly picked Yakima Valley-grown Centennial hops used to create it. Dark straw in color with light caramel malt tones, 2015 Fresh Hop resembles an aggressively dry-hopped IPA, but packing the punch and flavor only fresh hops can deliver. In addition to Centennial hops, this beer features locally grown Simcoe, Amarillo, Cascade and experimental hops, helping this popular beer achieve its bright and delightfully bitter flavor. Available in 22-ounce bottles and on draft, this beer will only be available for a limited time.
One of Seattle beloved food trucks, Mesob at The Curb, will be on site Friday from 4:00 – 7:00. For more information, follow Two Beers Brewing on Facebook, Twitter and Instagram, or visit www.twobeersbrewery.com.


The Queen Of The World tells a story about a new sales rep being taken around his region by his sales manager. They end up out in the sticks, and pull into a diner for lunch. A bunch of farmers are at a nearby table, dressed in overalls and John Deere hats.
The new sales manager sniffs at the spectacle. "Man, they they sure look like a bunch of dumb hicks," he said.
His boss smiles. "You see that field across the road?"
The young guy says he does. "How much fertilizer do you think that would need for a corn crop?" The young man doesn't know.
"If you wanted to plant soybeans, how many plants per acre would give you the best yield?" Again, the young man doesn't know.
"When should you plant to minimize the risk of late frost while maximizing the growing season?" Again, nothing.
"Well, if you go over and ask those 'dumb hicks', every one of them can give you the answer."
And if you go to your local County Fair, you can see all the computer monitors in the tractors and combines. Oh, and the latest GPS farming tech. Just sayin'.
Little Mike showed once again that the intelligence of the political class can be pretty stupid. The Re-Elect Trump 2020 Campaign thanks him for his support.
Education, n. That which discloses to the wise and disguises from the foolish their lack of understanding.
- Ambrose Bierce


Intensive Agronomy, Diversification of Crops in the 19th Century: The Case Study on the Household Cultivation of Park Family in Matjil Village Yechŏn County Kyˇongsang Province
In the 19th century the crop rotation method practiced in Chosŏn proved greatly superior to that practiced in the same age in Europe. By contrast as time passed the marginal productivity of labor for dry field crops declined. Increasingly additional labor was needed to produce the same quantity of grain. The increase in the productivity of land in the late Chosŏn period was not based on increased use of fertilizer, but rather from the increased use of labor. The increase in varieties and the small-scale of production that marked the diversification of dry-field farming was not done for the purpose of send goods to market but rather for self-consumption. After cultivating small quantities of a variety of crop according to a plan for consumption, if any surplus remained farmers took it to market for sale. In this way the trends in development of agriculture in the late Chosŏn may be summarized as intensification marked by increased productivity of land and decreased productivity of labor, and the diversification of agricultural crops grown for the purpose of self-consumption.
4. 일손의 분산과 노약자 활용
다각화(Diversification of Crops),
근경(Sequential Double Cropping),


The Art´s Acre is an initiative of the schloss bröllin e.V., originating from the collaboration with the artist Anneli Ketterer. The field at the gates of Schloss Bröllins Krieg Production area for vegetables until 1990, here are 25 years of wild herbs and grasses. In 2015/16, with the support of the farmers from Fahrenwalde, the land was reclaimed by Anneli Ketterer as a studio for artists of all sports. Work with the theme soil / earth, present yourself on the field in Mecklenburg-Vorpommern with LandArt, sculpture, installation or performing arts.
The Art´s Acre is not only a spatial transition of the field in the artist's residence, but also socio-political and ecological topics that are in nature, rural exodus, exploitation of raw materials, or future nutritional deficiency. The Art´sAcre in Bröllin is a long-term project that is being developed together with the people of the region. These include the village communities of the municipality Fahrenwalde and all within live also schools and day-care centers. The project seeks support from environmental and farming associations, science, politics, education and culture.
more information: http://decrustate.net/


Benefits of Hiring a Seed Specialist.
In farming, what you get is determined by what you sow. Therefore, you should always purpose to have the best seeds in order to get a handsome outcome. Do not think that determining the great seeds from the perfect ones is something you can do with your naked eye. You will have a better chance of getting the right seeds if you consult a deed specialist. Seed specialists are always conducting research on how to improve the seeds which are already available so that farmers will have a better shot at producing healthy and high produce. If you have limited land to till, you need to make sure that whatever you are planting will have high yields. To be a farmer who makes some handsome amount from the activity, every seeds that you plant should give the highest yield possible. In order to make sure that you get the highest yield, you should have a seed specialist guide you in choosing the right seeds.
Seed engineers have access to a lot of research on various seeds and you can benefit from this information if you engage them in your farming. Applying information which is backed by research is less likely to fail you because the scientists work with experts in conducting the research and analysing the findings which is why you can trust the information. In the event that you have questions concerning the seeds of the sowing process, you will get answers from a person who has a lot of knowledge in the sector and not just anyone who thinks that he has the information.
When you are working with a seed specialist, you also have the benefit of getting expert advice on various issues from the networks he or she has already built. You should think through the money and time you will lose if you approach farming blindly. It does not take a lot of money to hire a seed specialist but you will even make more if you let their expertise help you. With a good relationship, the specialist like Caudill Seed will always let you know about the advancements which have been made and if you are to keep up a high yield, you need to depend on every research you can get. Farming is a noble activity and it also has great profits if done right.


Supporting our local growers here in Sonoma County.
California and Sonoma County in particular is bountiful when it comes to fresh produce. We are lucky to be surrounded by local growers and family farms, they are our friends, neighbors and part of our community here at Fircrest Market and we support them in every way we can. All the while bringing you the best in fresh produce our area has to offer.
Orange juice fresh squeezed in our store
Every week we take the freshest oranges we can find and squeeze some of the best fresh orange juice. This is some of the freshest orange juice you can find without all the added stuff you don’t want. Just like nature intended.
The Seasons best
Our selection of fresh seasonal produce.
With each Season comes a bounty full of unique flavors that bring back memories of enjoying sweet strawberries in the Summer sun. The warm feeling of gathering around a table with your friends and family with a bowl full of hearty winter vegetables in a soup your mother used to make. Our buyers at Fricrest Market make sure you have all the Seasons best flavors to choose from.


- Paulo Ferreira 2022
- Coffee Commercial Manager
- Coordinadora de America Latina y el Caribe (CLAC)
- Global Session
- "The Future of Coffee must be Sustainable for the Planet and Fair for the Farmer"
Paulo Ferreira: 3rd Generation in coffee production in Brazil started as a coffee farmer and still hold the 2ha land of Organic and Fairtrade Coffee in South Minas in partnership with his father and brother.
As his family lives in the main coffee producer region in Brazil, Paulo got a degree in Coffee Technology from the Federal Institute of South Minas and began working in the commercial department of a Fairtrade and Organic cooperative in Brazil.
There as commercial manager, he was able to implement projects focused on increasing highest quality coffee production and in direct exportation. Paulo has a Master’s degree in International Trade Management and Human Resources and Team Development.
Since February 2022, he joined CLAC Fairtrade as Coffee Commercial Manager to support and help all Latin American Fairtrade producers to promote their personal histories, excellent fieldwork and amazing coffees.


Farmers markets are a great place for people looking for fresh food that’s grown locally. It can be hard to know where fruit and veggies are from if you buy them at the supermarket, and many places don’t always provide the freshest produce. Farmers markets solve this problem, and they also provide a way for you to support the local economy. Check out the Fairgrounds Farmers Market this fall for a wide selection of food and crafts.
At Cronic Nissan, we’re proud to be a part of the Griffin, GA community, and often attend the Griffin Famers Market. The event is held at the Kiwanis Fairgrounds throughout the year, and the next one will be held on Friday, September 1st from 9am to 1pm, according to Eventbrite. There’s constantly a call for new vendors, so there’s always new produce and products at the market.
You can find local products at the farmers market like Baker’s Bark spice rub, Grazing Here grass-fed beef burgers, fresh pumpkins, honey, jelly, and jam. Pecans are ever-popular, though you can get virtually any veggie or fruit you might need for your next dinner recipe.
Visiting the Fairgrounds Farmers Market is just one way you can support your community. Hop in the car and explore what the area has to offer.


The Champion family has owned vineyards on the commune of Bergères Les Vertus for several generations. This land, located on the world-famous Côte des Blancs, is officially classified as “premier cru”.
The grapes grown are almost exclusively of the Chardonnay variety, which give the champagne its elegance and delicacy.
Patricia and Denis Champion currently manage the estate, which includes 7.2 hectares of vineyards 5.5 hectares of which are located in Bergères Les Vertus. The wines produced are made at the famous Coopérative Union Champagne.
By combining our wines with other wine makers on other “premier cru” and “grand cru” land, we are able to ensure a constant quality of undated Denis Champion and Champion-Rifflard champagnes as well as a more extensive range of wines made from blends including Pinot Noir grapes, a variety that is little grown on our own estate.


Trichomes, found on the surface of cannabis and hemp flowers, are minuscule hair-like structures. They play a vital role in the production and storage of cannabinoids, terpenes, and other compounds within the plant. Beyond their chemical significance, trichomes serve multiple purposes such as safeguarding the plant, defending against predators, and facilitating the production of essential oils and resins.
Trichomes are responsible for the frosty and sparkling appearance of cannabis and hemp flowers. They come in various sizes, shapes, and densities, with the highest concentration found on the calyxes, leaves, and stems. The flower’s bracts, small leaf-like structures that protect the reproductive parts of the plant, contain an especially rich concentration of trichomes.
Trichomes are responsible for synthesizing and storing cannabinoids such as CBD (cannabidiol) and THC (tetrahydrocannabinol), as well as terpenes and flavonoids. These compounds play a vital role in the plant’s interactions with the environment, attracting pollinators and deterring potential predators.
When hemp flowers are harvested and processed, the presence of trichomes becomes crucial in determining the flower’s cannabinoid and terpene content, as well as its overall quality. These trichomes contain many of the compounds responsible for the potential therapeutic effects and aromatic qualities of hemp. As a result, consumers often seek out trichome-rich flowers to fully experience the benefits and unique characteristics of different hemp strains.


You may not have noticed but London’s grape harvest has just finished. And I think, unintentionally, I was the last link in the chain yesterday as I delivered my pathetic 2.3 kilograms of grapes from our garden (enough to qualify for one bottle of wine) bang on the deadline of 12pm at the collecting point, Unit 13 on Merton Industrial Estate. This is where Chateau Tooting, the crowd-sourced vineyard, collects a gallimaufry of grapes from gardens and allotments around London and turns them into wine at an established winery. Last year it was Bolney Estate which did a fantastic job of transmuting these grapes into what turned our to be a very palatable wine. This year they went to the well regarded Halfpenny Green vineyard in Shropshire for processing.
Chateau Tooting is still London’s largest winemaker but not for much longer. Co-founder Richard Sharp hopes this year to produce more than the 662 bottles recorded last year but soon he will be overtaken by Forty Hall, the admirable community start-up in Enfield, which has a vineyard stretching to ten acres within sight of Canary Wharf and the Shard. (see above) They produced their first bottles earlier this year but there was only a limited quantity which was reserved for volunteers and patrons (such as me). It looks like there will only be a limited quantity next year as well from this year’s harvest (just collected) because they have wisely been doing some savage pruning in order to strengthen the harvest in later years when they plan to produce 10,000 bottles. If you want to help or become a sponsor you can do so here.
Paul Olding makes Olding Manor on his allotment in Lewisham. His yield is down this year to 60 to 70 bottles but for a good reason. He has fulfilled his bucolic dream by buying an acre and a half of pasture land in east Sussex near the Kent boarder on which to create a proper vineyard. This will take a few years to establish itself and meanwhile he is able to be a bit more choosy about his grapes and experiment a bit with different yeasts.
Alex Smith who grows grapes along a wall in the yard of his factory at Kings Cross (below) lost his entire output to the birds. He said: “I bought netting and covered the vines but the starlings still found a way in”
Tony Hibbert, who runs Clocktower vineyard in west London reports that the weather was perfect for grapes and about 6 weeks ahead of last year, so he is hoping for a much higher sugar content. However powdery mildew has destroyed a large proportion of the grapes, but those which survived he is leaving for as long as it takes to reach 12% alcohol without adding sugar.
Marko Bojcun has planted a vineyard called Hawkwood in Chingford to grow for the local community making wine for dozens of households who bring their grapes to him.
All of this is small stuff compared with the resurgence of vineyards in the rest of England and Wales. But from small acorns . . .