POST GRADUATE SCHOOL

INDIAN AGRICULTURAL RESEARCH INSTITUTE

NEW DELHI- 110012

OUTLINE OF RESEARCH WORK

1. Name of the student : Gajendra Ramesh Rathod

2. Roll No : 10520

3. Discipline : Plant Physiology

4. M.Sc. /M.Tech. /Ph.D. : Ph.D.

5. Date of joining of P.G School : 1st August, 2014

6. Major Field : Plant Physiology

7. Minor Field : 1. Molecular Biology and Biotechnology

2. Genetics

8. Title of the Thesis : Physiological analysis of root traits and architecture for adaptability and yield under water deficit stress in Wheat (Triticum Aestivum L.)

9. OBJECTIVES:

1. Evaluation of root traits associated with deeper rooting and adaptability to moisture stress.

2. To study the physiological and molecular aspects of deeper rooting for adaptability to water-deficit stress.

10. PREVIOUS WORK DONE

Role of root traits in adaptation to water deficit stress in wheat

Wheat is one of the first domesticated food crops and is widely cultivated cereal crop,

with over 225 mha area. It contributes about 20% of the dietary calories and proteins

worldwide (Shiferaw et al., 2013). About 50% of the approximately 230 mha under wheat

cultivation annually in the world is frequently affected by drought (Reynolds et. al., 2005).

Enhancing the yields under water-limited environments is challenged by the complex and

quantitative nature of the drought response and the complex genomic constitution of wheat.

In rainfed agriculture, potential evaporation and transpiration often exceed precipitation

(Nagarajan, 2009), which limits water availability for crop production. Globally, drought is

the second most (7.5%) extensive hazard after flooding (11%) (Nagarajan, 2009). For crop

production under water-limiting conditions, plants need to withstand in this conditions for the

longer duration. The magnitude of yield decline due to drought is depend upon critical stage

and severity of drought. Plant biomass, which is a crucial parameter, decreases under drought

stress in spring wheat (Wang et al., 2005). The same outcomes were observed in previous

studies in wheat and other crops (Wang et al., 2005; Watson et al., 1952; Sudhakar 1993). In

winter wheat, the yield was decreased or changed under drought and, in contrast, the water

use efficiency was boosted (Xue et al., 2006 ). In the vegetative phase decrease in stomatal

opening occurs, which alters CO2 exchange, leaf expansion, and stem elongation (Hale et al.,

1987) and conclusively results in decreased crop growth. Due to determinate growth habit of

cereals the reproductive phase is considered as the most critical stage (Saini and Westgate,

2000), because any damage done at that stage cannot be restored. As Crop production under

water limited condition is the product of transpiration (T), transpiration efficiency (TE) and

harvest index (HI) (Passioura, 1977). In this context grain yield is linked to post-anthesis

transpiration or crop water used (Van Oosterom et al., 2011). Therefore, identification of

traits associated with water utilization of crops is important to increase crop productivity

under water limited condition.

One of the important traits for adaptability to water limited conditions is the root system

architecture which can directly influence the capacity of roots to extract soil moisture and

maintain yields. Roots continue to grow to find water, but the growth of aboveground organs

is limited in drought stress conditions. This type of differential growth response behavior of

shoots and roots to drought is an adaptation to arid conditions (Sharp et al., 1989; Spollen et

al., 1993). Root-to-shoot ratio rises under drought conditions to facilitate water absorption

(Morgan et al., 1984; Nicholas et al., 1998) which are connected to the ABA content of roots

and shoots (Rane et al., 2001). The growth rate of wheat roots was decreased effectively

under moderate and high drought conditions (Noctor et al., 1998). The possible ways to

ensure future food needs of an increasing world population involves the development of crop

varieties which needs less water, more tolerant to drought and which may avoid water stress

by deeper rooting. Introducing the deep-rooting characteristic into shallow-rooting cultivars

is considered as one of the most promising breeding strategies.

Importance of deeper rooting

Root is the hidden half of the plant and it performs many essential functions such as

nutrient and water uptake, storage organ, anchoring to the soil and by interacting with the

beneficial organisms in the rhizosphere and many more which are unknown yet.

Increasing population and changing climate patterns along with constraints in availability of

water are challenges for survival of mankind on the Earth. To face these challenges we

requires the strategy to use this water cautiously, for this we need crop cultivars and

agricultural system that can remain productive in erratic weather patterns and which are

capable of more efficient in resource capture from soil. Drought resistance in crops is

required to ensure the global food security.

Plant strategies for deeper rooting

Among the many strategies for coping and adapting to water deficit stress, deeper

rooting is one of the important plant morphological characteristics. Deep rooting is an

important root system architecture type which may help plants to avoid drought-induced

stress by extracting water from deep soil layers (Uga et al., 2013). The first green revolution

was achieved due to reduced shoot height in 1970s but now we need to find and evaluate

root phenotypes and depth genes which can extract water from deeper soil layers which

leads to increase in survival chances of crop. The intensity of deep rooting depends on two

main components (i) the root gravitropic response component and (ii) the primary growth of

the roots based on assimilates supply.

Root growth in relation to carbon supply

The control of the rate of root growth depends on the acquisition of carbon by roots

(Farrar and Jones, 2000). Both the supply of assimilate to roots and their utilization is

required for root growth. Most studies point to the control of acquisition of carbon by roots

by both shoot and root (i.e. shared control) (Farrar and Jones, 2000). Root vigour depends on

photo assimilate and water allocation to root tips for growth (Boyer et al., 2010). Faster root

growth depends on processes within the root apex that determine cell division and expansion

(Sharp et al., 1989). The assimilate produced from photosynthesis are partitioned to starch

and sucrose. It was observed that of the total carbohydrate received by the roots over a 24hr

period, relatively more material was apparently obtained during the day. Thus, relatively

higher root growth was related to partitioning of assimilates to sucrose production during the

day. Sucrose phosphate synthase plays a major role in photosynthetic sucrose formation

(Huber et al., 1983). Carbon partitioning toward Sucrose is increased in numerous higher

plant species in response to osmotic stress (Huber et al., 1996). Osmotic-stress activation of

sucrose-phosphate synthase in some plants is achieved by Protein phosphorylation (Toroser

et al., 1997). Sucrose transport and distribution are key steps in plant stress resistance as it is

involved in regulation of plant growth and plant stress tolerance. Sucrose transporter

AtSUC9 mediates abiotic stress resistance by low sucrose level regulating sucrose

distribution and ABA accumulation (Jia et al., 2015). Utilization of sucrose as a source of

carbon and energy requires cleavage enzymes e.g. invertases which play an important role in

early sucrose partitioning, plant development and metabolic fluxes (Tang et al., 1999).

Sucrose synthase is also a major biomarker for sink strength in potato (Zrenner et al., 1995).

Protein kinases are the key metabolic regulators of carbon partitioning. A protein

kinase that plays a key role in the global control of plant carbon metabolism is SnRK1

(sucrose non-fermenting-1-related protein kinase 1), so-called because of its homology and

functional similarity with sucrose non-fermenting 1 (SNF1) of yeast (Halford et al. 2003).

The carbohydrate allocation in plants is also regulated by osmoregulated processes

(e.g.osmosensitive kinases, OsmK). The decrease of SnRK1 and increase of OsmK results in

partitioning of carbon towards sucrose to supply growing sink tissue (Pokhilko et al., 2015).

Transgenic Arabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt

and freezing stresses. The transgenic showed a decreased rate of water loss, enhanced higher

relative water content, strengthened cell membrane stability, improved photosynthesis

potential and significantly increased osmotic potential, enhanced osmotic potential, growth

and development under both normal and stress condition (Mao et al., 2010). Nine of the 10

SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating

an important role in osmotic signaling (Boudsocq et al., 2004). Multiple SnRK2–type protein

kinases were detectable in the root extracts and showed differential response to ABA

treatment (Coello et al., 2012). Overexpression of TaSnRK 2.3 improved root system and

significantly enhanced tolerance to drought, salt and freezing stress (Tian et al., 2013).

Inactivation of SnRK 2.6 alter the steady-state levels of leaf soluble sugar and starch under

well-watered conditions (Zheng et al., 2010). Rapid changes in sink–source relations in the

annual Nicotiana attenuata after simulated herbivore attacks increased the allocation of

sugars to roots. This herbivore-induced response is regulated by the β-subunit of a SnRK1

(GAL83) transcripts which were rapidly down-regulated in source leaves after herbivore

attack and, when silenced, increase assimilate transport to roots (Schwachtje et al., 2006).

Several members of the SnRK2 family of plant-specific kinases are also key players in ABA

hormone signaling and can be activated by exogenous ABA in the absence of osmotic stress.

However, other SnRK2s cannot be activated by ABA but can still be rapidly activated by

osmotic stress. How activation of these latter SnRK2s is connected to osmotic stress is not

known (Boudsocq et al., 2007 ; Haswell and Verslues, 2015). However, it is important to

note that these SnRK2 play important role in the regulation of metabolism and their

overexpression / transgenics showed higher sucrose synthesis under stress.

The plant hormone auxin functions as a signalling molecule and a driver of growth

and developmental processes which are regulated by the availability of free sugars which

depend on the activity of sucrose cleavage enzymes e.g. invertases and sucrose synthase. IAA

homeostasis responds rapidly to alterations in the carbohydrate levels in A. thaliana (Siranen

et al., 2012). Arabidopsis auxin receptor tir1 and response mutants, axr2, axr3 and slr1 not

only display a defect in glucose-induced the change in root length, root hair elongation and

lateral root, suggesting glucose effects on plant root growth and development are mediated by

auxin signalling components (Mishra et al., 2009).

Root gravitropic response in relation to deeper rooting

The physiological process of root gravitropism comprises gravity perception, signal

transmission, growth response and the re-establishment of normal growth (Sato et al., 2015).

The downward growth of roots or root gravitropic response can be quantified / phenotyped in

terms of root growth angle (Manschadi et al., 2006). Asymmetric root growth and downward

bending of the root in response to gravity helps in improvement of drought avoidance due to

probability of deeper rooting. Higher expression of DRO1 increases the root growth angle

whereby roots grow in a more downward direction (Uga et al., 2013). They also observed

that DRO1 is negatively regulated by auxin. Auxin plays an important role in the gravitropic

response of the roots (Sato et al., 2015).

Regulation of auxin transport is related to the orientation of roots (Sato et al., 2015).

The auxin transport is regulated by the expression of PIN proteins which is also dependent on

the cellular redox status (Considine and Foyer, 2014). However, the post-transcriptional

redox mechanism is still uncharacterized. The cellular redox regulator Glutathione has been

found to play role in root growth as it is involved in auxin transport (Koprivova et al., 2010).

The plastid-localized GR2 is essential for root growth and root apical meristem (RAM)

maintenance (Yu et al., 2013).

11. PROGRAM OF RESEARCH WORK

Hypothesis based on the previous work done

Sucrose is mainly utilized for osmotic adjustment and plant growth (shoot and root).

The surplus amount of sucrose is used for root growth under water limited conditions. Thus,

the control of the rate of root growth depends on the acquisition of carbon by roots. Both the

supply of assimilate to roots and their utilization is required for root growth. But the

utilization of this surplus sucrose as a source of carbon and energy requires proper activity of

cleavage enzymes such as Invertase and sucrose synthase in the roots. Various SnRKs and

osmosensitive kinases play a key role in the global control of plant carbon metabolism and

allocation under normal and osmotic stress conditions. Decrease in SnRK1 activity leads to

carbon partitioning toward Sucrose. Carbon partitioning toward sucrose is increased in

response to osmotic stress which may be due to activation of various SnRK2s. Presently it is

not known that how activation of these SnRK2s and osmosensitive kinases like OsmK is

connected to osmotic stress? The availability of free sugars, probably due to sucrose cleaving

enzymes, determines homeostasis and transport of the plant hormone Auxin. Auxin transport

regulates the gravitropic response of the roots via PIN proteins which is also dependent on

the redox status of roots.

By combining these studies we can get a further clear picture on the role of root

gravitropic response component and the primary growth of the roots based on assimilate

supply in regulation of deeper rooting. We hypothesize that the variability in deeper rooting

and adaptability to moisture stress in wheat depends on the shared control of sucrose

partitioning by shoot and root, root redox status and morphophysiological traits.

Therefore, in view of the importance of such studies further efforts are needed to

understand the physiological basis of diverse aspects of root traits such as - genotypic

differences in root distribution in the soil profile, rates of water uptake after anthesis and

cooler canopies, genotypic variability in root angle, proportion of seminal and nodal roots,

role of fine roots in relation to enhanced surface for absorption, root/shoot ratio, total root

length, surface area, early root vigour, root-shoot vasculature in relation to water transport,

hydraulic properties of roots and anatomical characteristics. Keeping in view the above facts,

the present investigation is proposed to be carried out with following objectives to evaluate

root traits associated with deeper rooting and adaptability to moisture stress and to investigate

the physiological and molecular aspects of deeper rooting.

Experiment No. 1 (related to objective 1): Evaluation of variability in root architecture

and growth in relation to deeper rooting.

1a. To evaluate seminal root angle by agar gel method.

In this experiment, the wheat root geotropic response will be measured in 100 wheat

genotypes. The root geotropic response will be measured in terms of the seminal root

angle in wheat seedlings in the agar gel plate. Thereafter, these genotypes will be

classified with respect to the seminal root angle - from very narrow to very wide angle.

From these genotypes, 20 genotypes will be selected from different angle classes for the

actual root growth upto anthesis in 1m PVC pipes.

1b. To evaluate deeper rooting and root growth in wheat genotypes in 1m PVC pipes.

The 20 genotypes selected from above experiment will be studied for actual root growth

and deeper rooting in 1m PVC pipes with three replications. The plants will be grown

under normal and water stress condition imposed during booting. Water stress will be

evaluated by measuring the Relative Water Content (RWC). Stress will be started from

booting stage and watering will be provided when plants show leaf rolling and it is not

recovered. The roots will be washed at anthesis and root morphological traits will be

studied. The root morphological traits include – maximum root length, total root length,

root distribution according to different diameter classes, root surface area, root volume

and root biomass etc. in different soil horizons (0-30, 30-60, 60-90) to find the

variability in relation to deeper rooting. Shoot biomass, leaf area and no. of leaves will

also be measured in plants grown in PVC pipes. Based on the root growth of PVC pipes

further selection of six contrasting wheat genotypes will be made to understand the

physiological and molecular aspects of deeper rooting as indicated in objective no.2.

Experiment No. 2:

To examine the role of sugar status in leaves and roots, sucrose utilization in roots, its

relationship with SnRKs in relation to dry matter partitioning to roots for deeper

rooting under moisture stress.

To study the role of root redox and ROS status in wheat genotypes showing

differential deeper rooting behavior under moisture stress.

Observations to be recorded in Experiment No. 2:

(a) Leaf

1. Photosynthesis and stomatal conductance under normal and stress conditions.

2. Total sugars, reducing sugars, non reducing sugars

3. Sucrose Phosphate Synthase activity

4. SnRK1 and SnRK 2.4 expression

(b) Root

1. Total sugars, reducing sugars, non reducing sugars

2. Invertase activity

3. Sucrose synthase activity

4. SnRK1 and 2.4 expressions under control and stress condition

5. Root OSCA1 expression

6. Root redox status

7. Root ROS content

(c) Yield and yield components

12. METHODOLOGY

(i) Root seminal angle (Manschadi et al., 2006)

(ii) Root morphological traits using root image analyzer: WinRhizo (Arsenault et al.,

1995)

(iii) Relative Water Content (Barr and Weatherley 1962)

(iv)Photosynthesis and stomatal conductance using Infrared Gas Analyzer

(v) Total sugars, reducing sugars, non reducing sugars (McReady 1950, Nelson 1944,

Somogyi 1952, Hodge and Hofreiter 1962)

(vi)Sucrose Phosphate Synthase activity (Huber and Israel 1982 )

(vii) SnRK1 and SnRK 2.4 expression (Mao et al., 2010)

(viii) Invertase activity ( Long et al., 1975, Nelson 1944)

(ix)Sucrose synthase activity (Huber and Israel 1982)

(x) Root OSCA1 expression (Yuan. et al., 2014)

(xi)Redox status- roots (Zhang et al., 1996, Loggini et al., 1999 )

(xii) ROS status- roots (Dunand et al., 2007)

Statistical Analysis of physiological and biochemical data will be performed as per

observations Panse and Sukhatme 1978.

13. FACILITIES REQUIRED AND THEIR AVAILABILITY

The present study will be carried out at the Division of Plant Physiology, along with facilities

at National Research center on Plant Biotechnology and division of Genetics, IARI, New

Delhi 110012.

Student Date :

Recommended by:

1. Advisory committee:

Name Signatures

Chairman Dr. Rakesh Pandey, Division of Plant Physiology, IARI

Co-chairman Dr. Vijay Paul, Division of Plant Physiology, IARI

Member Dr. C. Viswanathan, Division of Plant Physiology, IARI

Member Dr. P.K. Mondal, NRCPB

Member Dr Neelu Jain, Division of Genetics, IARI

14. Whether Radioactivity is involved in the proposed research work? No

If yes,

i. Whether radioactivity badge has obtained or applied for? NA

ii. Whether the laboratory in which the work has to be carried out is approved for

Radioactivity work? NA

Certified that the ORW of the student has been formulated and finalised in accordance with

the procedure prescribed in Para 8.14.2 of the PG School calendar.

2. Professor ………………………… Date: ……………..

3. Head of the division …………………………. Date: ……………..

4. Approved by Dean, P.G. Schoo

………………………….. Date: ………………

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