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1. Root system architecture : opportunity for improving drought tolerance in crops AKSHAY S. SAKHARE SUDHIR KUMAR DIVISION OF PLANT PHYSIOLOGY ICAR - INDIAN AGRICULTURAL RESEARCH INSTITUTE

2. Roots are marvelously multitalented organs Act as fingers and mouth Act as arms and legs Stomach Propagators

3. Root system architecture (RSA) is defined as the spatial and temporal arrangement of roots There are two types of roots tap roots and adventitious roots Tap roots Adventitious roots

4. Root system architecture Genes Environment Hormones

5. Genes To date, 675 QTLs related to root traits such as root length, thickness, volume and distribution have been detected. Several transcription factor gene families have been shown to influence abiotic stress tolerance (NAC, WRKY, AP2/ERF and DREB). In rice, DEEPER ROOTING 1 (DRO1) gene has been shown to control deep root exploration, which increases yield under drought conditions. Genomic loci that confer advantageous RSA and yield

6. Soil properties influence RSA Soil properties such as density and particle size influence RSA Soil compaction leads to shorter roots with large diameter and so the resulting RSA is shallow and narrow Root elongation is more affected by mechanical and physical properties than chemical properties of soil. Full understanding of how the physical properties of soil impact RSA will allow for the development of crops that can thrive in different soil types. RSA responds to nutrient heterogeneity Environment

7. The world population is expected to reach 9 billion by the middle of the twenty-first century. Rice, a staple food for nearly half of the world’s population, is particularly susceptible to drought-induced stress owing to its shallow rooting relative to other cereal crops. To improve drought avoidance in rice, identifying genes influencing deep rooting and clarifying their effects on drought avoidance is necessary

8. Two distinct accessions in a preliminary screen for natural variation in root system architecture IR64 Kinandang Patong (KP) Dro1-NIL They developed a near-isogenic line homozygous for the KP allele of DRO1 (DRO1-kp) in the IR64 genetic background (Dro1-NIL) to compare the effects of DRO1-kp and the IR64 allele of DRO1 (DRO1-ir) on deep rooting under field conditions. The maximum root depth of Dro1-NIL plants was more than twice that of IR64 plants

9. Steps in the monolith sampling method Driving the monolith sampler into the ground Pulling the monolith sampler from the ground. Dividing the soil monolith into 12 parts. Study of the root growth of Dro1-NIL and IR64 plants useing monolith sampling method Each soil monolith was divided into 12 blocks (A to L), and root dry weight in each block was measured. Condition of root samples obtained by using the sampler

10. Dro1-NIL plants had more roots distributed in deeper soil layers than IR64 plants Effect of DRO1 on root dry weight in different soil layers in an upland field

11. Effect of DRO1 on shoot traits in an upland field Dro1-NIL plants did not show any significant differences from IR64 plants in total-root dry weight or in shoot traits DRO1-kp allele changed only the root distribution in the soil

12. Deep rooting in cereal consists of the root growth angle and maximum root length The root growth angle ( θrga ) of each plant was determined by measuring the angle between the soil surface (horizontal line) and the shallowest nodal root. Mean root growth angle of IR64 and Dro1-NIL at around 30 days after sowing.

13. Effect of DRO1 on root and shoot morphological traits in a hydroponic system Dro1-NIL plants showed slight differences in root length from IR64 plants But no marked differences in other root and shoot traits These results suggest that DRO1 mainly influences root growth angle

14. Positional cloning of DRO1 By comparing the sequence of ORF Os09g0439800 (LOC_Os09g26840.1) in the IR64 and KP accessions a single 1-bp deletion within exon 4 in IR64 was identified, which resulted in the introduction of a premature stop codon . High-resolution mapping narrowed the candidate region to a 6.0-kb segment (a) Candidate region of DRO1 locus reported previously. CEN, centromere. (b) Physical map of region around DRO1 gene on chromosome 9. (c) Graphical genotypes and phenotypes of BC3F2 plants containing recombination between marker loci CAPS05 and INDEL09. Blue, homozygous for IR64 allele; red, homozygous for Kinandang Patong (KP) allele; yellow, heterozygous. IR64-homo and KP-homo are BC3F2 plants homozygous for alleles from IR64 and KP, respectively, in the entire candidate region.

15. By transforming an 8.7-kb genomic fragment of KP containing the candidate gene into IR64 they found that the ratio of deep rooting was increased as compared to IR64 transformed with the vector control Ratio of deep rooting of transgenic IR64 (T2 plants) containing a single copy of the 8.7-kb DRO1 genomic fragment from KP ( KPg -sc) or the empty vector (V). Basket assay of representative transgenic plants containing vector control and a single copy of the 8.7-kb DRO1 genomic fragment from KP in the IR64 genetic background (20 plants in each line). Scale bars, 10 cm. Ratio of deep rooting as the number of roots that penetrated the lower part of the mesh (≥50° from the horizontal, centered on the stem of the rice plant) divided by the total number of roots that penetrated the whole mesh. A larger value for the ratio of deep rooting means that a greater proportion of the roots grew downward.

16. Comparison of deduced amino acid sequences between DRO1 and its homologs in other plants DRO1-kp, DRO1 of rice cv. Kinandang Patong . DRO1-ir, DRO1 of rice cv. IR64. DRO1-np, DRO1 of rice cv. Nipponbare . Non-rice sequences are XM_003578131.1 ( Brachypodium distachyon ), scaffold543596 18.8 ( Hordeum vulgare ), XM_002462405 (Sorghum bicolor), EU969762 ( Zea mays ), XM_002516205.1 ( Ricinus communis ), LOC100808146 ( Glycine max), LOC100249804 ( Vitis vinifera ), LOC101207004 ( Cucumis sativus ), At1g72490 (Arabidopsis thaliana), XM_002326096.1 ( Populus trichocarpa ), and MTR_8g021200 ( Medicago truncatula ). Amino acid residues of DRO1 homologs identical to those in DRO1-kp (gray shading) are indicated by black shading. Amino acid residues identical across all accessions are boxed in yellow rectangles. The red arrowhead at the bottom of the figure indicates the position of the 1-bp deletion in IR64 relative to Kinandang Patong . Black bars above the sequences indicate putative N- myristoylation sites predicted by Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). There is no similarity between the predicted protein product of DRO1 and other known proteins. Genes found in monocots had higher homology to DRO1 than genes found in dicots. And

17. Determination of the subcellular localization of the DRO1 protein, they introduced a construct encoding a DRO1-kp–YFP (yellow fluorescent protein) or DRO1-ir–YFP fusion protein into rice protoplasts DR01-kp::YFP DR01-ir::YFP YFP (Control) YFP Merge DIC Subcellular localization of DRO1 in rice protoplasts transformed with CaMV35S::DRO1-kp::YFP or CaMV35S::DRO1-ir::YFP via electroporation. In rice protoplasts transformed with CaMV35S::DRO1-kp::YFP, YFP fluorescence was visible in the cell membrane under a confocal laser scanning microscope. In protoplasts transformed instead with CaMV35S::DRO1-ir::YFP or CaMV35S::YFP (control), YFP fluorescence was visible in the cell nucleus and cytoplasm as well. Left, YFP fluorescence; center, merged YFP and (differential image contrast) DIC images; right, DIC alone. Scale bar, 10 μ m. In fluorescence study DRO1-kp–YFP was localized to the plasma membrane Which suggests that DRO1 is associated with a membrane protein or with the plasma membrane. In contrast, the DRO1-ir::YFP was localized to the nucleus and cytoplasm as well as to the plasma membrane

18. To understand the mechanisms underlying the DRO1-controlled phenotype, they investigated root growth angle in the seminal roots of IR64, Dro1-NIL and several DRO1 transgenic lines Effect of DRO1 on root growth angle and root gravitropic curvature Root growth angle ( θrga ) of IR64 and Dro1-NIL plants 2 d after sowing in agarose gel. Scale bars, 1 cm. Distribution of root growth angle in IR64 and Dro1-NIL plants. IR64, vector control lines and RNA interference ( RNAi ) knockdown lines had a smaller root growth angle and higher variation in this trait than Dro1-NIL and the DRO1-kp transgenic lines, suggesting that DRO1-ir and RNAi knockdown lines have less gravitropic curvature than DRO1-kp lines

19. Gravitropic curvature in seminal roots of IR64 and Dro1-NIL plants. Seedlings were grown on agarose for 2 d and then rotated either 60° or 90° from the original vertical axis for 4 h. θiar , inclination angle of root , representing the angle between the root and the horizontal axis immediately after rotation; θrac , root angle of curvature after rotation. Asterisks indicate the positions of root tips at the start of rotation. Yellow arrows indicate the direction of gravitational force. Scale bars, 1 cm. Time course of the root angle of curvature after horizontal rotation in IR64 and Dro1-NIL plants. Seedlings were kept in the dark after rotation, and curvature was measured once per hour for 12 h. As gravitropism is one of the most important factors determining root growth angle, they performed a time course study of root gravitropic curvature This experiment showed that the roots of Dro1-NIL plants responded more abruptly to rotation from the normal vertical axis to the horizontal axis than those of IR64 plants These results suggest that DRO1-kp confers greater gravitropic curvature than DRO1-ir and that the increased gravitropic response in Dro1-NIL plants relative to IR64 plants was caused by their gain of DRO1 function.

20. Expression analysis of DRO1 DRO1 expression in several shoot and root tissues. Expression data for DRO1 were normalized to the expression of a ubiquitin gene. Samples of the root tip, the middle part of the root, the basal part of the shoot (1-cm sample from the bottom of the shoot, including the meristems of adventitious root primordia ), the middle part of the shoot and the leaf blade were taken from plants grown in baskets 30 d after sowing. Pulvinus and spikelet samples were taken 54 d after sowing and 1 d before flowering, respectively. The expression of DRO1 in Dro1-NIL plants was more than double that in IR64 plants in the root tip and the basal part of the shoot Relationship between DRO1 expression level and the ratio of deep rooting 42 d after sowing. V, vector control; KPg -sc and KPg -mc, transformed plants containing a single copy or multiple copies, respectively, of the DRO1 genomic fragment from KP (DRO1-kp); RNAi , plants containing an RNAi knockdown cassette for DRO1. They examined the relationship between DRO1 expression and root growth angle using DRO1 transgenic plants. A positive relationship between DRO1 expression and the ratio of deep rooting, demonstrats that enhancement of DRO1 expression can increase the root growth angle, resulting in deeper rooting.

21. In situ hybridization of DRO1 mRNA in the root tip of a Dro1-NIL plant. Scale bars, 200 μm . Expression patterns of DRO1 in tissues of Dro1-NIL

22. Expression patterns of DRO1 in tissues of Dro1-NIL a b c d (a) In situ hybridization with a DRO1 -specific sense probe on a longitudinal section of the root tip of Dro1-NIL at 30 days after sowing. (b–d) In situ hybridization of DRO1 near the basal part of the shoot at 4 days after sowing. (b, d) Hybridization with an antisense probe; (c) hybridization with a sense probe. (b, c) Longitudinal section; (d) transverse section. DRO1 was expressed around the root apical meristem in the root tip and crown root primordia in the basal part of the shoot, suggesting that DRO1 is expressed mainly around root meristem tissues. DRO1 was also expressed in the spikelet, but no marked phenotypic difference was observed between the spikelets of IR64 and Dro1-NIL plants

23. The phytohormone auxin has a key role in root gravitropism, so they examined the effect of auxin on DRO1 expression Expression of DRO1 and OsIAA20 in response to auxin. Seedling root tips of IR64 and Dro1-NIL plants were treated with 10 μM 2,4-D. Effects of auxin (2,4-D) and cycloheximide (CHX) on DRO1 transcript levels in Dro1-NIL. Five-day-old seedlings of Dro1-NIL were treated with 10 μM 2,4-D and/or 10 μM CHX. DRO1 expression was substantially decreased within 30 min of treatment with auxin (2,4-D), whereas the expression of OsIAA20 (an auxin-response marker gene18) was increased DRO1 expression was inhibited by auxin, even in the presence of cycloheximide , suggesting that de novo protein synthesis is not required for DRO1 repression by auxin Molecular characterization of DRO1

24. Binding of recombinant OsARF1 protein to AuxRE sequences in the DRO1 promoter region. The arrowhead indicates the shifted band. The locations of fragments 1, 1m, 2 and 3 are shown in. Fragment 1m contained a single-nucleotide substitution (G to A) relative to fragment 1. Competitor was unlabeled fragment 1 (or fragment 1m, where designated). This finding suggests that DRO1 is an early-auxin-response gene that might be directly regulated by ARFs in the auxin signaling pathway. Electrophoretic mobility shift assays was performed to examine whether ARF proteins would interact with AuxREs The recombinant rice ARF protein OsARF1/OsARF23 bound to the RE1-containing fragment 1.

25. Expression profiling of auxin-related genes in IR64 and Dro1-NIL roots at 3 h after auxin treatment Microarray analysis based on a two-color system with auxin-treated samples (3 h; Cy5) and pre-treated samples (0 h; Cy3) was performed . FDR, false discovery rate. The log ratio of signal intensity (log2 Cy5/Cy3) was used to construct the heat map. The criterion for inclusion was FDR < 0.05 for at least one of the two samples (IR64 and Dro1-NIL). The data are means of three independent biological replicates, and the values for genes represented by multiple probes were averaged. Green vertical bar, auxin biosynthesis genes46; orange, signaling genes46; blue, polar transport genes28–30,47. The ARF protein used in the electrophoretic mobility-shift assay is OsARF23. Expression patterns in response to auxin were similar in the two lines for most of the auxin-related genes tested, suggests that DRO1 is not involved in the regulation of these genes. Transcriptome analysis of auxin-related genes in the root tips of IR64 and Dro1-NIL plants before and after treatment with auxin.

26. Root bending of representative plants (24 plants in each line) 1.5 h after horizontal rotation. Red and black dashed lines indicate outer perimeters from quiescent centers (asterisks) to elongation zone (large arrowheads) on the upper and lower side of roots, respectively. Scale bars, 200 μm . Comparison of the cell length of the epidermis in the elongation zone on the upper and lower sides of the root 1.5 h after horizontal rotation. In Dro1-NIL plants, the lower side of horizontal roots showed a shorter outer perimeter than the upper side 1.5 h after horizontal rotation When horizontal roots of IR64 and Dro1-NIL plants (rotated 90° from the original vertical axis) Results suggest that auxin localization on the lower side of the root tip after horizontal rotation represses DRO1 expression in the distal elongation zone DEZ, resulting in decreased cell elongation relative to the upper side. This process may contribute to asymmetric growth, leading to root gravitropic curvature, although further analysis is needed to clarify how DRO1 controls cell elongation.

27. To investigate the effect of DRO1 on drought avoidance, they compared the grain yields of IR64 and Dro1-NIL plants under upland field conditions with no drought, moderate drought or severe drought Effect of DRO1 on the response to drought-induced stress Time course of the moisture level at different soil depths in treatments with no, moderate and severe drought-induced stress under a rainout shelter at CIAT. A soil moisture probe was used to record observations around noon each day in 18 access tubes installed across the experimental area. Black and white arrowheads indicate the time points at which drought conditions were initiated and terminated, respectively. Top view of responses of IR64 and Dro1-NIL plants to 27 d of severe drought-induced stress. Percentage of filled grain and grain weight for IR64 and Dro1-NIL plants grown under conditions of no, moderate and severe drought-induced stress. Moderate drought significantly reduced the grain weight per plant in IR64 plants, whereas Dro1-NIL plants had almost the same grain weight per plant Under severe drought, the percentage of filled grain in IR64 plants was nearly zero, whereas that in Dro1-NIL plants was >30% In the no-drought plots, both lines had similar yields. Filled grain (%) Grain wt / plant (g)

28. In a second drought experiment, they measured the photosynthetic capability, root distribution and yield potential of both lines under more severe drought conditions than in the severe-drought plots of the first experiment Physiological and morphological differences between IR64 and Dro1-NIL under drought stress conditions Visible and thermal images of IR64 and Dro1-NIL at 30 days after the start of drought stress Side and top views of the block at 37 days after the start of drought stress. By then, all plants of Dro1-NIL showed panicle emergence. In IR64, few heading plants were observed and leaf rolling had occurred. Top view Side view Visible image Thermal image The leaf temperature of Dro1-NIL was lower than that of IR64 Under severe drought, physiological damage such as leaf wilting and delayed flowering was more prominent in IR64 than in Dro1-NIL plants

29. Effect of DRO1 on photosynthesis, yield performance, and vertical root distribution under drought stress Soil monolith sampling showed that Dro1-NIL plants had more root mass in deeper soil than IR64 plants. Dro1-NIL plants had lower leaf temperature and higher photosynthetic capability than IR64 plants. The drought-induced flowering delay was shorter in Dro1-NIL plants than in IR64 plants. Consequently, the final grain weight per plant was significantly higher in Dro1-NIL plants than in IR64 plants. Difference between canopy temperature of Dro1-NIL and IR64 ( C) Photosysnthesis rate ( μ molCO 2 m -2 s -1 ) Stomatal conductance (molm -2 s -1 ) Days to 50 % flowering Total above ground dry weight/plant (g) No. of panicles/plant Grain weight/plant (g) Grain weight/panicle (g) 1000-grain weight (g) Total root dry weight (g)

30. To clarify the relationship between the root’s ability to access soil water and drought avoidance, they evaluated the grain yields of IR64 and Dro1-NIL plants in a third drought experiment, under a drought condition in which capillary flow of water from lower to upper soil layers was blocked by a layer of gravel Experimental conditions in an artificial drought stress system in which access to soil water is limited Cross-sectional diagram of physical environments in drought avoidance assay. Blue arrows represent capillary water flow in the soil. Blue dashed lines indicate plastic nets between gravel and soil layers. View of assay system in the greenhouse before the start of drought stress. Time course of soil water potential in the artificial drought conditions

31. Effect of DRO1 on drought avoidance in an artificial drought system in which access to soil water is limited Cross-sections of assay system (top panels) and views from above gravel layer (bottom panels) in the drought plot at harvest time. Yellow asterisks in upper right image indicate roots that had elongated through the gravel layer. Yellow arrowheads in lower right image indicate roots penetrating into the gravel layer. Some roots of Dro1-NIL, but not IR64, grew through the gravel layer. Top views of rice plants at 28 to 49 days after the start of drought stress. At later stages, many IR64 plants in the drought plot had leaf rolling, and some had leaf wilting. Some roots of Dro1-NIL plants grew through the gravel into the lower soil layer, but those of IR64 plants did not.

32. Effect of drought stress on shoot- and yield-related traits measured in IR64 and Dro1-NIL Dro1-NIL plants thus had higher grain yield than IR64 plants These results confirm that, under drought conditions, deeper rooting by DRO1 facilitates better photosynthesis and grain filling, resulting in higher yield.

33. Conclusion The DRO1-kp allele enables rice to produce more grains under drought-induced stress. Under non-drought conditions, these plants show no yield penalty because DRO1 alters only root growth angle and does not decrease either shoot or root biomass. Other economically important monocots, such as maize, contain DRO1 homologs that may be useful for enhancing drought avoidance in other crops. Control of root system architecture may contribute to drought avoidance in crops.