Comparison of Phenotype of Plants Expressing Non-polar Nip5; 1 Mutant With Wild-type in Response to Boric Acid Stress.

Boron is a very essential element nutrientfor growth and improved fertility of plants. Its deficiency and toxicity affects the plant development. Globally, most areas suffers from this problems of boron deficiency and toxicity [V.M. Shorrocks, 1997].

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Boron toxicity is mainly seen in arid and semi-arid areas while its deficiency are commonly observed in areas with increased rainfall [K, Miwa, and Fujiwara, T., et al, 2010].

The dry nature of arid and semi-arid areas concentrate boron while the rain, decreases the boron concentration by leaching.
This world-wide agricultural problem prompted the research on boron transporter for development of a more tolerant crops that will enable efficient regulation and adequacy of boron within the plant tissue.

Plant absorbs boron mainly as boric acid/borate. Boron becomes either toxic or deficient when its concentration exceeds or decrease the normal range, 30µMB. NIP5; 1, a major intrinsic protein of the subgroup nodulin 26-like intrinsic protein, first discovered from Soybean [J. Danielson and U. Johanson, 2009],were responsible for efficient boron uptake under boron limitation. This protein could be seen at the outer plasma membrane root of the plant cells. The regulatory nature of this protein were co-studied with BOR1, a boron exporter under boron limitation, with a GFP tagged Nip5; 1 and BOR1. In boron deficient condition, NIP5; 1 imports boron from the soil side into the epidermal tissue, the BOR1 exports the boron into the stele. A mutant line, nip5; 1-1, a T DNA insertion of Col-O (WT) background, were studied and found that it lacks the boron absorbing potential under boron limitation due to variation in the gene that encodes the NIP5;1 [Takano et al, 2006]. But on the contrary, its corresponding wild type (Col-0) develops very well on boron limitation due to presence of NIP5; 1.

AIM
The aim of this research to examinetheeffect of the polarity of nip5; 1 on roots growth of different wild and mutant type of Arabidopsis thaliana: polar GFP fused NIP5; 1(WT) and 3 non-polar independent mutant (Nip5; 1 without UTR GFPNip5; 1(3 threonine residues /3 alanine residues).

OBJECTIVES
To achieve this aim, we compare the different phenotypes of Arabidopsis thaliana on different boron concentration: boron toxicity (100µMB) and limitation (1µMB and 0.1µMB). The growth of roots and level of plant development confirms whether a plant absorbs boron or not. On fluorescence visualization, the WT GFP is polar while that of mutant is non-polar. Also we observe the tolerant and sensitivity nature of plant expressing NIP5; 1 protein on both concentrations. This observation enables a biotechnologist to choose the actual tolerant plant for a particular boric acid stress that would be suitable for growth at a particular point in time.

 

CHAPTER TWO
LITERATURE REVIEW

Aquaporin
Aquaporin are integral membrane protein from a larger family of Major Intrinsic Protein (MIP) that form pores in the membrane of biological cells. They could be localized at the intracellular and plasma membranes and function as protein channels that facilitate the transport of water and/or small neutral solutes (Urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide).
In plant physiology, water and some essential plant nutrients are absorbed by the root, where it passes from the cortex into the vascular tissues. Plant studies revealed that the movement of water to and from the xylem and phloem may occur by one of, or in some cases, by the combination of the following pathways: Apoplastic pathway, the symplastic pathway and the transcellular pathway. [Ingela J. et al, 2000].

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In Symplaspic pathway, water is transported in the cytoplasmic continuum of adjacent cells connected through plasmadesmata, while the transcellular pathway involves transport across the plasma membrane and the vacuolar membrane [Ingela J. et al, 2000]. In a typical Arabidopsis root, the Casparian strip of the endodermal cells, acting as a barrier, severely restricts the radial water movement in the apoplast. In microscopy study, Casparian strip seprates the Outer and Inner plasma membrane of GFP tagged Nip5; 1 and BOR1 respectively [Yoshinari, A.et al, 2012].
Aquaporin could be divided into four main homologous subfamilies:

i. The plasma membrane Intrinsic Protein (PIP) that are mainly abundant in the plasma membrane-hence the PIP. It has two phylogenic subgroup: PIP 1 and PIP 2.

ii. The tonoplast intrinsic protein (TIP) which is mainly localized at the vacuolar membrane. It has five phylogenic subgroup: TIP1, TIP2 TIP3 TIP4 and TIP5

iii. The Nodulin-26like intrinsic protein (NIP). This named after Soybean (Glycine max). They are as well present in non-leguminous. It has several phylogenic subgroup.Such as NIP 1s, NIP 2s, NIP 3s, NIP 4s, NIP 5s and NIP like MIP. For Arabidopsis, only two subgroup exists: subgroup I and II [J. Danielson and U. Johanson, 2009]

The small basic intrinsic protein (SIP).
Current studies revealed three additional sub families of Major intrinsic protein in Physcomitrella patens-GlpF-like intrinsic protein (GIP), hybrid intrinsic protein and X intrinsic protein. In Arabidopsis study, plant aquaporin shows a high multiplicity of isoform with 35 homologs in Arabidopsis thaliana; there are 13homologs for PIP, 10 homologs for TIP, 9 homologs for NIP and 3 homologs for SIP[Christophe M. et al, 2008]. Also 33 and 36 homologs of aquaporin could be seen in Oryza sativa [Christophe M. et al, 2008 and Sakurai et al, 2005], and Zea mays [Chaumont F. et al, 2001] respectively.

Functions of aquaporin
In a typical plant, aquaporin contributes to plant growth and development by transporting water into the tissues. Water flows along various paths: (i) the apoplastic path, that is, within the cell wall continuum, (ii) the symplastic route through cytoplasmic continuities and plasmodesmata, and (iii) the transcellular path across cell membranes (mainly plasma membranes).[Ingela J. et al, 2000 and Chritophe M. et al, 2008]. Aquaporin also facilitates the transpiration, tissue desiccation and imbibition, tissue expansion in plants. Study showed that Aquaporin could function in Nitrogen fixation, CO2 transport and carbon metabolism and especially nutrient uptake in plant such as NIP5; 1 functions as a boric acid channel for boron uptake and as well essential for plant growth under low boron stress. [Takano J. et al, 2006]. Two T- DNA insertions were developed and the nip 5; 1 mutant were sensitive to low boron condition.

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Structure of aquaporin

Structurally, aquaporin proteins consists of six transmembrane alpha helices arranged in a right handed bundle, with the amino and the carboxyl termini located on the cytoplasmic surface of the membrane. There are also five interhelical loop region (A-E) that form the extracellular and cytoplasmic vestibules

This structure depicts a typical plant aquaporin consisting of both the amino and carboxylic terminal, the six transmembrane and the five interhelical loop and the NPA motif in B and E.(modified from Training and Mobility Research network project).
Studies suggested that the loop B and E contains the asparagine, proline and alanine (NPA) motif.

Boron deficiency in plant
1. Boron is an essential micronutrient, needed for plant growth and development. Its deficiency affects the plant propagation by hampering vegetative and reproductive growth which could lead to tripartite abnormalities in plant. The three abnormalities are inhibition of the cell expansion, reduced fertility and death of meristem [H. Marschner, 1995]. Among all the micronutrient deficient disorder, these are the most widespread abnormalities in all parts of the world and causes large losses in crop quantity and quality [V.M. Shorrocks, 1997], which could lead to global food insecurity and hunger. Commonly, Boron exists as Boric acid (H3BO3) and a minute increase or decrease on the required concentration leads to toxicity or deficiency. Boron in plants may be in water-soluble and insoluble form. The amount of water-soluble B fluctuates with the quantity of B supplied, while insoluble B does not. [Matoh, T., et al, 2001]. The solubility nature of soil boron could be easily leached by intense rainfall. Regions with intense rainfall are more prone to boron deficiency [K, Miwa, and Fujiwara, T., et al, 2010], arid and semi-arid regions suffers from boron deficiency. A very good hypothesis is the wheat commonly grown in arid northern part of Nigeria. Wheat thrives well in this region because of its boron-toxicity tolerant nature. Some of the symptoms of boron deficiency depends on species. The boron visible symptoms of these three crops (Grape, wheat and Sorghum) commonly used biotechnology industry are:For grape, the visible symptoms are “Hen & Chick” symptom, dead main shoots. Wheat; Distorted heads and chlorosis of leaves. Sorghum; Leaves are narrow and have a gray appearance with watery, transparent stripes. Seed heads are not filled.

Major intrinsic protein NIP5; 1 is essential for boron uptake under boron limitation
NIP5; 1 is Major intrinsic protein from the subfamily of nodulin 26-like intrinsic protein of the phylogenic sub group II, for Arabidopsis thaliana. NIP was found in Soybean Glycine maxNip1:1 (GmNip1:1). Study suggested that NIP is localized to theperibacteroid membrane of rot nodules. NIP has several phylogenic subgroups and specific among various species. Phylogenic study of Arabidopsis thaliana NIPs (AtNIP) classified NIPs into two phylogenic subgroups: Subgroup I (AtNIP1; 1, AtNIP1; 2, AtNIP2; 1, AtNIP3; 1, At4; 1 and At4; 2) and Subgroup II (At7; 1, At5; 1 and At6 😉 [J. Danielson and U. Johanson, 2009].
NIP5; 1 has been found to play a crucial role in boron uptake under low boron condition [Takano J. et al, 2006]. This was achieved by development of two T- DNA insertion lines (nip5; 1-1 and nip5; 1-2). This two mutant lines showed decreased boron uptake under low boron condition when compared with the wild line. Under boron limitation, NIP5; 1, a boric acid channel imports boron into the epidermal tissue and BOR1, a boric acid transporter exports boron for xylem loading of the plant tissues [Takano J. et al, 2005].The NIP5; 1 and BOR1 localized on the outer plasma membrane (soil side) and inner plasma membrane respectively, when visualized with a confocal microscopy for Green Fluorescence Protein fused NIP5; 1 and BOR1 respectively.

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Plant root structure
Root is the non-flowering part of the plant that typically lies below the surface of the soil. It functions as anchorage part of a plant, Water and inorganic nutrients absorbent, storage of foods and nutrients and encourages vegetative reproduction. The first part of the root that emerges first during germination is the radicle or primary root. In a typical cross-section of mature root, it comprises of Cortex, epidermis and the stele. The stele contains endodermis, pericycle, xylem, phloem, cambium and cortex. The phloem tissues conducts products of photosynthesis from leaves throughout plant including down to the roots. The xylem on the other hand, conducts water and minerals up from the roots through the plant. The longitudinal cross-section of a root comprises the meristematic zone (consisting of the root tip meristem and the root cap), the zone of elongation where new cell enlarges and the zone of maturation or differentiation.

Arabidopsis thaliana.
Arabidopsis thaliana is a small flowering plant native to Eurasia of the family, Brassicaeae that are used as an important toolbox in most molecular biology research. Arabidopsis thaliana has many advantages for genome analysis, including a short generation time, small sizeand large number of offspring, efficient transformation while utilizing Agrobacterium tumefaciens, availability of large number of mutant lines and a relatively small nuclear genome [AGI, 2000 and National institute of health].It has 5 chromosome and a genome, approximately 135megabasepair that was first completed by AGI in 2000. Carl Linnaeus renamed this plant (Pilosella siliquosa) Arabidopsis thaliana in honor of the discoverer, Johannes Thal in 1842. Study showed that is often between 2 to 25cm in height, with a basal rosette of leaves that are 0.8 to 3.5cm long and up to 1cm wide. Information about Arabidopsis and newly developed lines are contained in Arabidopsis Information Resource (TAIR) or Nottingham Arabidopsis stock center (NAS) database

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