WUSCHEL-related homeobox gene WOX11 increases rice drought resistance by controlling root hair formation and root system development

ABSTRACT

Roots are essential organs for anchoring plants, exploring and exploiting soil resources, and establishing plant-microorganisms communities in vascular plants. Rice has a complex root system architecture consisting of several root types, including primary roots, lateral roots, and crown roots. Crown roots constitute the major part of the rice root system and play important roles during the growing period. Recently, we have refined a mechanism that involves ERF3/WOX11 interaction is required to regulate the expression of genes in the cytokinin signaling pathway during the different stages of crown roots development in rice. In this study, we further analyzed the root phenotypes of WOX11 transgenic plants and revealed that WOX11 also acts in controlling root hair development and enhancing rice drought resistance, in addition to its roles in regulating crown root and lateral root development. Based on this new finding, we proposed the mechanism of that WOX11 is involved in drought resistance by modulating rice root system development.

Plant root organogenesis is regulated by a complex network that involves many genetic and environmental factors.^1-3 Organization and cell differentiation processes in taproot development have been well characterized in Arabidopsis.¹ Nevertheless, cellular origin and the mechanisms underlying the development of the fibrous root system in cereal crops remain unclear. Rice, one of the most important food crops worldwide, has a fibrous root system including primary, crown and lateral roots, and root hairs. Impressive progress has recently been made in rice regarding crown roots formation and which advanced our understanding of the regulatory network involved in the development of the monocot root system.^2-4 However, the mechanism underlying rice root hair developmental program in rice is largely unknown.

Drought is one of the most severe abiotic factors that affect crop productivity worldwide. A cost-effective solution for sustainable crop production in water-limiting areas is the development of crop cultivars with high drought tolerance. Thus, identification of the germplasm with enhanced drought tolerance and determination of the tolerance mechanism are quite imperative. When plants are exposed to drought stress, root hairs form an important and large surface area for efficient absorption of water and minerals. Although the mechanisms of root hair development have been extensively studied in Arabidopsis⁵ and the molecular information on root hair formation in monocot species is studied by the identification of some genes in maize,⁶ rice,^7,8 and barley,^9-11 no information is available in regard of the relevance of root hair growth and response to drought stress.

Our previous study demonstrated that WOX11 plays an important role in the emergence and growth of crown roots.¹² Recently, we further showed that WOX11 is involved in crown root development through interaction with ERF3 to repress type-A RR gene, RR2, affecting the homeostasis of cytokinin signaling in crown roots.¹³ WOX11 promoter activity is detected mainly in the tips and cell division zones of the primary and lateral roots, with the strongest activity in the epidermal cell layer.¹² Based on these results, we hypothesized that WOX11 might also be implicated in controlling rice root epidermis differentiation and root hair development.

To further verify whether WOX11 was involved in root hair formation, Surface-sterilized seeds of wox11 mutant, WOX11 over-expression transgenic plants (OxWOX11) and wild type (WT) were germinated on Murashige and Skoog (MS) medium in a square plate (12 cm × 12 cm). Five days later, we examined the root hair phenotype and found that wox11 mutant developed very short and limited root hairs, while WOX11 overexpression transgenic plants (OxWOX11) dramatically increased root hair length and number compared to wild type (Fig. 1A). These results suggested that WOX11 might regulate the root hair growth in rice. To validate the effect of root system architecture on drought resistance, wox11 mutant and wild type (WT) plants were subjected to drought stress treatment (soil water content ≈28%) at the booting stage in the fields. During the stress, wox11 mutant showed severer rolling-leaf phenotype compared to wild type (Fig. 2A). Water loss rate of detached leaves from wox11 mutant and wild type (WT) plants at 5-leaf stage were measured at the indicated time point. In agreement with the phenotype, the detached leaves from wox11 mutant lost water more quickly (t-test, P< 0.05) than wild type (WT) plants (Fig. 2B). This finding clearly indicated that wox11 mutant was more sensitive to drought stress.

Figure 1.

Root hairs and root system phenotypes of wild type (WT), WOX11 transgenic plants. A. Root hairs phenotypes of wild type (WT), wox11 and WOX11 over-expressing plants (OxWOX11). Surface-sterilized rice seeds were germinated and grown on Murashige and Skoog (MS) medium in a square plate (12 cm × 12 cm). One-week-old plant roots were observed. PR, primary root; RH, root hairs. Bars = 2 mm. B. Root system phenotypes of wild type (WT) and wox11 plants with two tillers in the field. Bar = 5 cm.

Figure 2.

Analysis of phenotype under drought stress and relative water loss rates in the detached leaves. A. Phenotypes of Wild type (WT) and wox11 mutant after drought stress (soil water content ≈28%) in the fields. B. Water loss rate assay of detached leaves from wox11 mutant and wild type (WT) at the 5-leaf-stage. Data represent the mean ±SE (n = 3). *P < 0.05, **P < 0.01, t-test.

Drought-induced leaf-rolling was impaired in gid1

Root hairs are slender projections originating from the epidermal cells, and they have important function in water and nutrient uptake.¹⁴ Development of root hairs in plants is affected by environmental factors such as drought, heavy metal and interactions with pathogenic and soil microorganisms.^15-19 Water stress caused by 20% PEG 6000 greatly induced root hair development in drought tolerant Tibetan wild barley XZ5.¹⁰ Our results also showed that wox11 mutant displayed not only shorter and less root hairs but also less root biomass than wild type at vegetative growth stage in the fields (Fig. 1B). Additionally, expression of WOX11 was quickly up-regulated after drought stress induction.²⁰ wox11 mutant was more sensitive to drought stress (Fig. 2). Taken together, all of these results indicated that WOX11 was involved in drought resistance by modulating rice root system and root hair development.

In addition to WOX11, another rice WUSCHEL-related homeobox gene, OsWOX3A, regulates root hair development by influencing auxin-transport gene expression.⁷ Although WOX11 expression can be induced by auxin,¹² the expression level of OsWOX3A does not change in WOX11 transgenic plants. These results suggest that mechanism of root hair development regulated by WOX11 might be different from that of OsWOX3A. Further studies are needed to clarify the relationship between these two genes in order to further understand the molecular regulatory mechanism of root hair development in rice.

Based on the results of our studies and previous reports,^4,12,13,20 we propose a model to interpret the drought resistance mechanism regulated by WOX11 (Fig. 3). Plant hormones and drought stress induce WOX11 expression. During crown root elongation, WOX11-ERF3 interaction enhances WOX11-mediated crown roots growth. Meanwhile, WOX11 is also involved in lateral root primordium initiation, crown roots elongation, and root epidermis cell to differentiate into root hairs, which ultimately increase rice root biomass, and facilitate water uptake, leading to drought resistance. To summarize, all of these results highlight the roles of WOX11 in the drought tolerance of rice, indicating that WOX11 could be exploited in biotechnology to improve drought resistance via manipulating WOX11-promoted root hair growth, lateral root initiation and crown root elongation in rice.

Figure 3.

Proposed WOX11 regulatory network involved in drought resistance by controlling rice root system development. Plant hormones and drought stress induce WOX11 expression. WOX11-ERF3 interaction enhances WOX11-mediated RR2 repression, which activates cytokinin response and leads to crown root elongation. Meanwhile, WOX11 is also involved in lateral root primodium initiation and root hair formation, leading to increased drought resistance. Arrows and a line ending with a trait represent the positive or negative regulatory action of one element of the network on another one, respectively. RR2, type-A RESPONSE REGULATOR2; WOX11, WUSCHEL-Related Homeobox 11; ERF3, ERE Response Factor 3.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31371468), the Program for New Century Excellent Talents in University (NCET-12-0863), and the Fundamental Research Funds for the Central Universities (2662015PY228).