Soluble invertase expression is an early target of drought stress during the critical, abortion-sensitive phase of young ovary development in maize

Abstract

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.

Figure 1

Development of relative available soil moisture over time for well-watered control plots and four drought treatments. The loamy sand soil had a capacity for available soil water of 100 to 130 mm. se shown (n = 3–4).

Figure 2

The effect of increasing drought stress on ovary ABA concentrations at five different stages of ear development. Legend as for Figure 1. F-tests and comparison of means for each stage was performed with the Gabriel-algorithm as described in caption for Table I (n = 3–4).

Figure 3

The effect of increasing drought stress on ovary dry mass (A), Suc (B), reducing sugars (C), and starch concentrations (D) at five different stages of ear development. Legend as for Figure 1. F-tests and comparison of means for each stage was performed with the Gabriel-algorithm as described in caption for Table I (n = 3–4). Aggregate statistical analyses for the entire period, where effects of dates were removed, showed that Suc concentration (B) was significantly higher under severe stress (t₃ and t₄) than in control (t_c) and moderate stress (t₁ and t₂); reducing sugars (C) were significantly lower under severe stress (t₃ and t₄) than under moderate stress (t₁ and t₂); and starch (D) was significantly higher under severe stress (t₃ and t₄) than in control (t_c) and moderate stress (t₁ and t₂).

Figure 4

The effect of increasing drought stress on soluble acid invertase activity (A) and insoluble acid invertase activity (B) at five different stages of ear development. A unit of enzymatic activity (U) equals 1 μmol of Suc hydrolyzed per minute under the assay conditions described in “Materials and Methods.” Legend as for Figure 1. F-tests and comparison of means for each stage were performed with the Gabriel-algorithm as described in caption for Table I (n = 3–4). Aggregate statistical analyses for the entire period showed that soluble acid invertase activity (A) was significantly lower under severe stress (t₃ and t₄) than in control (t_c) and moderate stress (t₁ and t₂). Paired t-tests showed that soluble acid invertase activity (A) was significantly higher (α ≤ 0.05) than insoluble acid invertase activity (B) on −6 d, −4 d, 0 d, and +3 d and significantly lower on +7 d.

Figure 5

The correlation between the hexose to Suc ratio (w/w) and soluble acid invertase activity. A unit of enzymatic activity (U) equals 1 μmol of Suc hydrolyzed per minute under the assay conditions described in “Materials and Methods.” Regression analysis was performed with the PROC REG procedure (SAS Institute Inc., 1988). *** denotes that the correlation is significant at the 0.1% level. Each point is the mean of three to four observations.

Figure 6

A, Gel blots showing the effect of increasingly severe drought stress treatments on accumulation of Ivr2 and Incw2 mRNA in maize ovaries from −6 d before to +7 d after pollination ([t_c] = control, [t₂] = moderate stress, and [t₄] = severe stress, as described below). The apparent size of both Ivr2 and Incw2 mRNA was about 2.1 kb. B, Relative levels of Ivr2 and Incw2 mRNA quantified from maize ovaries between −6 d before to +7 d after pollination based on two replicates (n = 2) of the following treatments: [t_c], control; [t₁], last irrigated July 28; [t₂], last irrigated July 17; [t₃], last irrigated July 03; and [t₄], last irrigated June 21). Shading of bars (left to right) indicates increasing severity of drought stress treatment. F-tests and comparison of means for each stage were performed with the Gabriel-algorithm as described for Table I. The statistical analyses showed that Ivr2 mRNA level was significantly lower under severe stress (t₃) than in control (t_c), both on +3 d and for the entire period as a whole. For RNA gel blots, 20 μg of total RNA was loaded into each lane, and density of ribosomal RNA bands was used as a loading control. These were uniform in all instances.

Figure 7

A, The correlation between soluble acid invertase activity and relative mRNA levels of Ivr2. B, The correlation between insoluble acid invertase and relative mRNA levels of Incw2. A unit of enzymatic activity (U) equals 1 μmol of Suc hydrolyzed per minute under the assay conditions described in “Materials and Methods.” Regression analysis was performed with the PROC REG procedure (SAS Institute Inc., 1988). *** denotes that the correlation is significant at the 0.1% level. Each point is the mean of two observations.

Figure 8

In situ localization of Ivr2 soluble invertase mRNAs in tissues of young maize kernels (NK-508) at +6 d post-pollination. Labeled Ivr2 mRNAs are evident in several areas of the maternal tissues that make up the maize kernel at this stage of development, with strongest signal in cells surrounding the tiny, newly fertilized embryo + endosperm (minute and off the field of view; see drawing to lower right) and in cells near vascular bundles of the pedicel. Signal proximal to the embryo + endosperm is localized in basal regions of the nucellus (maternal tissue filling the central kernel during early development), plus cells of the lower pericarp (ovary wall). Individual kernels were sampled as soon as pollinated ovaries could be visually distinguished from non-pollinated neighbors. Fixed and sectioned samples were probed with sense (control, top right) and antisense (in situ localization) RNA probes synthesized with digoxygenin-labeled UTP from a 576-bp Ivr2 fragment as described in the text.

Figure 9

In situ assay of acid invertase activity in kernel tissues during the first phase of development (+7 d post pollination) under well-watered conditions (A and B) or severe drought-stress (E and F), with each image paired to a parallel, serial section (C, D, G, and H) stained with toluidine blue for structural clarity. A, Whole-ovary view during the first phase of kernel development under well-watered conditions, with invertase activity shown as increasing from light-gold (throughout young ovary) to black (placental region immediately adjacent to nucellus [contiguous maternal tissues]). B, Detail of basal kernel regions, with filial generation to the top left (endosperm and tiny embryo), maternal tissues to the bottom right (nucellus and placental zone), and parallel structural information in subsequent panels. C and D, Toluidine blue staining of structural features for comparison to parallel sections in A and B, showing tissues in the zone of greatest invertase activity for very young kernels to be anastomosing vascular bundles with contiguous maternal parenchyma (phloem, nuclei, and cell walls are stained indigo, and xylem, turquoise). E, Whole-ovary view during the first phase of kernel development under severe drought stress, with invertase activity shown as gradations of gold (as for A). F, Detail of E. G and H, Toluidine blue staining of structural features for comparison with parallel sections in E and F, showing tissues in the zone of greatest invertase activity for very young kernels under drought stress to be much reduced areas of anastomosing vascular bundles with contiguous maternal parenchyma (phloem, nuclei, and cell walls are stained indigo, and xylem, turquoise). I, Negative control stained for acid invertase activity but without substrate (Suc) addition, well-watered treatment. J, Detail of I; artifactual staining of xylem may be a result of endogenous peroxidases involved in lignin biosynthesis. Longitudinal 5-μm methacrylate sections were obtained within 20 μm and stained alternatively by the two methods. Acid invertase activity was visualized using a coupled enzymatic reaction with Suc as substrate. Hydrogen peroxide produced by Glc-oxidase was used to oxidize DAB in a reaction catalyzed by horseradish-peroxidase, which formed a dark-brown/yellow DAB deposit at sites of acid invertase activity. Counterstaining with silveroxide and staining of adjacent sections with toluidine blue is described in the text. em, Embryo; en, endosperm; nu, nucellus; ph, phloem; va, vascular strand; and xy, xylem. Bars in A, C, E, G, and I = 500 μm. Bars in B, D, F, H, and J = 100 μm.