The absorption of water from humid air by grass embryos during germination

Abstract

Grass embryos possess structures that do not occur in any other flowering plants. Due to the specific embryo structure and position, grass embryo surfaces may be exposed to surrounding air under partial caryopsis-soil contact conditions, but whether caryopses of the grass family (Poaceae) can sense soil air humidity to initiate successful germination under partial caryopsis-soil contact conditions remain unknown. Here, we found that grass embryos have the unique ability to absorb water from atmospheric water vapor under partial caryopsis-soil contact conditions. To absorb atmospheric moisture, grass embryos developed profuse and highly elongated hairs on the embryo surface. These hairs, classically known as coleorhiza hairs, developed only on the embryo surface exposed to humid air, and submergence of the embryo surface inhibited their development. In addition to humid air-dependent development, almost all other developmental features of coleorhiza hairs were substantially different from root hairs. However, coleorhiza hair development was regulated by ROOTHAIRLESS 1. Besides the genetic control of coleorhiza hair development, we also identified how caryopses manage to keep the hairs turgid in natural open environments as the hairs were highly sensitive to dry air exposure. Moreover, we video-documented the regulation of developmental processes. The unique humid air-dependent coleorhiza hair development and their ability to absorb water from water vapor present in microsites or soil air give grasses advantages in germination and seedling establishment. Ultimately, coleorhiza hairs may have contributed to the ecological success of the grass family.

Figure 1

Humidity sensation and the development of coleorhiza hairs on the grass embryo surface. A, Setup of partial caryopsis–water contact conditions in the glued-caryopsis technique (see “Materials and methods” for other techniques). B, The effect of opposite caryopsis–water contact orientations (EIW and EIDA) on caryopses germination in low humidity condition (RH ∼55%) (n = 15). C, Water absorption pattern of EIDA- and EIW-oriented dehulled caryopses in low humidity condition (RH ∼55%) (n = 3). D, Change of water content of imbibed caryopses after placement in EIDA and EIW orientations in low humidity condition (RH ∼55%) (n = 3). E, The effect of EIHA and EIW orientations on caryopses germination in high humidity condition (RH > 98%) (n = 15). F, Water absorption pattern of EIDA and EIHA-oriented dehulled caryopses (n = 3). G, Time-lapse images of a germinating caryopsis under EIHA condition. H, Longitudinal section view of EIHA-oriented rice embryo before radicle or coleoptile emergence. I–N, The development of coleorhiza hairs under EIHA condition across rice sub-populations. I, Indica. J, Aus. K, Ashwina. L, Rayada. M, Aromatic. N, Japonica. O–R, The development of coleorhiza hairs in cultivated and wild species of Poaceae. O, Bluegrass (Poa pratensis). P, Barley (Hordeum vulgare). Q, Ryegrass (L. perenne). R, Wheat (T. aestivum). S–T, Difference between coleorhiza and root hairs on (S) hair elongation pattern (n = 3) and (T) length of hairs (n = 25). U, The unimpaired development of coleorhiza hairs in the embryo-removed caryopsis. V, The development of coleorhiza hairs on the outer epidermal surface. W, The ability of isolated coleorhiza, epiblast, and scales to develop coleorhiza hairs. X, The retention of the hairline despite manual extension of exposed area. Y, The development of profuse and highly elongated coleorhiza hairs in the primary rootless mutant, nrtp1 (For all representative images, n ≥ 3; (B), (E), and (T), Mann–Whitney test; (C), (D), (F), and (S), Wilcoxon matched-pairs signed-rank test; NS, not significant; **P < 0.01; ***P < 0.001 and all error bars show ±sd, all scale bars = 1 mm.).

Figure 2

RHL1 promoter-driven GUS and GFP expression. A–C, RHL1 promoter-driven GUS expression pattern. A, At the beginning of hull and caryopsis coat rupture. B, At the end of caryopsis coat rupture. C, After caryopses germination. D–F, RHL1 promoter-driven GFP expression pattern shown in three panel sets (bright field, BF; fluorescent, F, and merged, M). D, At the end of caryopsis coat rupture. E, Longitudinal section view of rice embryo before radicle and coleoptile protrusion. F, After caryopses germination. Arrow indicates the empty glume sheath. (Scale bars = 1 mm).

Figure 3

Comparative phenotypes of mutant and complemented/overexpression lines of RHL1. A, WT. B, rhl1 mutant, and (C) RHL-complemented/overexpression line after caryopses germination. D, Comparison of the length of coleorhiza hairs between WT and RHL1 complemented/overexpression line (n = 5). D, Mann–Whitney test; *P < 0.05, error bars show ±sd, scale bars = 1 mm.

Figure 4

Comparative density of hair cells between root and embryo surface. A, The representative bright field (BF), fluorescent (F), and merged (M) images of transgenic rice (RHL1:GFP) roots. B, Comparison of the density of hair cells between root and embryo surface. Root (adj.) and root (tip) indicate root adjacent to coleorhiza and root-tip, respectively (n = 10). C, The development of coleorhiza hairs in every cell of the exposed embryo surface. Arrow indicates the empty glume sheath. B, Mann–Whitney test, ***P < 0.001 and error bars show ±sd, scale bars = 1 mm.

Figure 5

The high sensitivity of coleorhiza hairs to relatively dry air exposure. A, Illustration of the method used for the estimation of shrivel/collapse with an AU on time-context relative rotation numbers of coleorhiza hairs. Drawings of the hair are not to scale, nor in the order and position of rotations. B, Shrivel/collapse of coleorhiza hairs (also see Supplemental Movie S2) (r stands for Pearson correlation coefficient), (n = 10 hairs).

Figure 6

The natural occurrence of high humidity inside soil cracks/microsites and decreasing night-time temperature facilitates coleorhiza hair development in natural environments. A, Soil cracks—common microsites in clay-rich floodplain. B, Diurnal temperature and humidity patterns of air inside a soil crack situated 5 cm below soil surface on a random day. Shaded area represents nighttime. C, Diurnal temperature and humidity patterns of air inside mimicked soil cracks (made of agar-blocks) (n = 7). D, The relationship between diurnal changes of RH with that of the air temperature inside microsites (r stands for Pearson correlation coefficient) (n = 45). E, The relationship between diurnal changes of temperature with the duration of saturated humidity hours of microsites (n = 45). F, The relationship between the decrease of night temperature with the duration of saturated humidity hours inside microsites (n = 45). G and H, Induced condensation under microscope mimicking microsites and decreasing night-time temperature conditions on (G), caryopsis surface and (H), coleorhiza hairs. (Scale bar = 500 µm). I, Droplet growth pattern (n = 3) (error bar ± sd).

Figure 7

The extent of embryo–water contact influences coleorhiza hair development. A, Schematic outline of the set-up of micromanipulated embryo–water contact conditions. (see Supplemental Movies S3, S4, S5, and S6). B, The effect of embryo–water contact on number of coleorhiza hairs. For box plots, the center line, tiny squares, limits, whiskers, and points correspond to the median, mean, upper and lower quartiles, 1.5× interquartile range and outliers, respectively. (n = 10). C, The effect of embryo–water contact on length of coleorhiza hairs (n = 10). D and E, Revalidation of the inhibition of coleorhiza hair development after submergence of embryo surface under (D) EICWW and (E) EUW conditions. (For representative images, n ≥ 2. B and C, Mann–Whitney test; ***P < 0.001, scale bars = 1 mm).

Figure 8

Absorption of atmospheric water vapor/dew accelerates germination and seedling establishment. A, Comparison of surface area with (+ CH) and without (− CH) coleorhiza hairs. (Mann–Whitney test; ***P < 0.001, n = 25, error bar ±s.d). B, The fate of dew drop condensed on tip of coleorhiza hair in steady temperature condition. Arrow indicates the direction of droplet movement. Number on each figure represents minutes. The time point of the highest droplet diameter was considered zero (0) minute. Scale bar = 100 µm (also see fast-motion Supplemental Movie S7). C, The accumulation of large dew drops on coleorhiza hairs after mimicked decreasing night-time temperature condition. Scale bar = 1 mm (also see fast-motion Supplemental Movie S8). D, The effect of two caryopsis–water contact conditions (EIHA and OEIHA) and two RHL1 genotypes (WT and rhl1 mutant) on caryopsis coat rupture to radicle emergence time. For box plot, the center line, tiny squares, limits, whiskers, and points correspond to the median, mean, upper and lower quartiles, 1.5× interquartile range and outliers, respectively (n = 5) (Also see Supplemental Movies S3, S4, S9, and S10). Two-way analysis of variance (caryopsis–water contact × RHL1) showed a significant main effect of caryopsis–water contact (F_{(1, 16)} = 78.60, P < 0.0001) and RHL1 (F_{(1, 16)} = 10.35, P = 0.0054) and a significant interaction between factors (F_{(1, 16)} = 5.904, P = 0.0273), followed by Turkey’s multiple comparison tests (WT EIHA versus rhl1 EIHA, P = 0.0052; WT OEIHA versus rhl1 OEIHA, P = 0.9432; WT EIHA versus WT OEIHA, P = 0.0017; rhl1 EIHA versus rhl1 OEIHA, P < 0.0001).