Lecture 7 - Nutrient Uptake in Relation to Plant Demand

NUTRIENT UPTAKE

IN RELATION TO PLANT DEMAND

LECTURE # 7

READING ASSIGNMENT

Rawat et al, (1999) At AMT1 gene expression and NH₄⁺ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels. The Plant J. 19: 143-152.

Tie-Bang Wang, Walter Gassmann, Francisco Rubio, Julian I. Schroeder, and Anthony D.M.Glass Rapid Up-Regulation of HKT1, a High-Affinity Potassium Transporter Gene, in Roots of Barley and Wheat following Withdrawal of Potassium Plant Physiol. 1998 118: 651-659.Wang, T.B., Gassmann

Marschner, H., Kirkby, E.A., and Engels,C. (1997) Importance of cycling and recycling of mineral nutrients within plants for growth and development. Bot. Acta. 110, 65-273.

LECTURE OUTLINE

1. Focus on mechanism : (reductionism) By focussing on biochemical mechanism we have been very successful in biology but we may miss the whole plant perspective. In this lecture we treat ion transport into roots in terms of "whole plant demand". This involves thinking about the needs of shoots and how regulation of root uptake responds to the needs of the shoot or the growing fruit, which may be some distance from the root.

2. Evidence for regulation of ion uptake

a. Hoagland (1930's) is always credited with discovering that plants starved of nutrients gave very high estimates of K⁺ or NO₃^- uptake compared to plants given adequate supplies of nutrients.

b. In fact it was Brezeale (1906) using wheat plants, who showed that the uptake of K⁺, NO₃^-, Ca²⁺, and Pi were each enhanced when the plant was deprived of that nutrient for 15 h. (See Table 1).

3. Mechanism of regulation: influx or efflux ?

a. Tracers reveal that influx is the main factor.

b. Concentration plots

c. Effects on V_max

d. Some molecular studies with HKT1 : This is described in the paper by Wang et al., listed as a reading assignment.

e. Response to withdrawal :cytoplasm or vacuole ?

f. The Nitrate Story : Read Crawford and Glass (1998)

4. Cycling of nutrients in the phloem.

Other problems :root to shoot transfer. A model based on recycling of nutrients in the phloem. Read :

Marschner, H., Kirkby, E.A., and Engels,C. (1997) Importance of cycling and recycling of mineral nutrients within plants for growth and development. Bot. Acta. 110, 65-273.

NUTRIENT UPTAKE

IN RELATION TO PLANT DEMAND

INTRODUCTION

Early research on ion uptake focused heavily on the mechanisms of ion uptake, especially the source of energy to drive active transport (thermodynamics of solute uptake) as well as the kinetics of ion uptake which provided the first evidence for a carrier-mediated transport. With the developments in biochemistry and molecular biology we have become even more focused at the reductionist level of understanding. Much less attention has been directed toward understanding the integration of ion uptake in whole plant function. For one thing reductionism is very attractive; it usually very productive and by comparison with whole plant studies much more definitive. In other words whole plant integration is TOUGH. Nevertheless, it is very important and extremely interesting to view the larger picture.

DYNAMIC NATURE OF ION UPTAKE

1. Regulation at the uptake step: CaSO₄-grown plants

In the 1930's and 40's Hoagland, made use of barley seedlings grown hydroponically for about 1 week in solutions containing only 0.5 mM CaSO₄ to measure the uptake of K⁺, NO₃^-, and other ions. At a time when measurements of ion uptake were generally made without the advantage of radiotracers, it was necessary to optimize uptake or be satisfied with experiments that lasted for days, and therefore suffered from lack of sensitivity. Hoagland observed that growing the plants this way compared to growing them in full nutrient-solution, considerably increased the rates of absorption of all ions except Ca²⁺ and SO₄^2-. Actually, the observation was recorded as early as 1906 by Brezeale, who demonstrated significant increases of nitrate, phosphate, calcium, and potassium uptake when these ions were removed from the media for 15 h (table 1). Thus, it would seem as though the plant adapts to the absence of a particular nutrient by increasing the capacity to absorb that particular nutrient.

2. Mechanisms of the increase of uptake

Net uptake might be regulated by increasing influx or decreasing efflux. The use of tracers, such as ⁴²K⁺, ³⁵SO₄^2- and ¹³NO₃^- and ¹³NH₄⁺ (Figs 1, 2, 3 of the class handout and Figs 1, 2a,and 2b) enabled us to distinguish influx from efflux.

Figure 2a

Figure 2b

The large adaptive changes evoked by removing a nutrient have always been accompanied by increased influx. By measuring the release of tracer from roots after a brief exposure to tracer, it has also been possible to estimate efflux. This flux appears to increase as tissue concentrations of the ion are increased. However, this effect is small compared to the huge effect on influx. We have measured the K_m and V_max values for influx using concentration plots. These show very clearly the effect of tissue composition on influx (Table 3 and Table 7.2 of the class handout and Tables 2 and 3 below).

Table 2 : V_maxand K_m for K⁺ influx in barley roots previously starved of K⁺, then resupplied with K⁺ for a period of 12 h. (From a study by Glass, 1976)

Duration of K⁺ resupply (h)	Root [K⁺]	K_m for K⁺Influx (mM)	V_max for K⁺Influx (mmol g^-1FW h^-1)
0	26.3	36	7.3
3	64.6	54	5.4
6	93.9	66	2.9
12	130.5	133	1.13

Table 3: Effect of growth with or without various nutrients on V_maxand K_m for influx of SO₄^2-, Pi, and Cl^-. (From Lee, 1982)

Nutrition prior to measuring influx	Ion influx measured	V_max (nmol g^-1FW h^-1)	K_m (mM)
+ SO₄^2-	³⁵SO₄^2-	53.4	13.9
- SO₄^2-	³⁵SO₄^2-	758	17.6
+Pi	³²Pi	257	6.6
-Pi	³²Pi	475	4.9
+Cl^-	³⁶Cl^-	1010	57.4
-Cl^-	³⁶Cl^-	2600	23.7

In all cases, the V_max for influx was reduced by withholding the ion in question. The simplest interpretation of this observation is that the number of carriers is increased by withholding the ion. However, only very recently has it been possible to obtain strong evidence for this hypothesis. Using the case of K⁺ withdrawal, we know that influx increases in the first hours after removing K⁺ (Fig 2a). We have obtained the HKT1 gene clone for the high affinity K⁺ from Julian Schroeder at San Diego. Our evidence shows that after 4 h without K⁺, levels of expression of this gene had increased substantially (See Fig. 3). Furthermore, although influx continued to increase up to 24h the level of mRNA for the HKT1 gene did not increase substantially after 12 h. The low affinity gene appears not to be upregulated by withholding K⁺. Both the uptake data of Leon Kochian and the molecular biology data of a French colleague (Lepetit) at Montpellier confirm this. We are preparing to examine the NH₄⁺ gene expression but there are no other genes available yet.

Figure 3

The last sentence (in italics) was written at this time last year. I left it in to show you how fast things can change in some areas. Since last year we have examined the AMT1 gene which codes for high affinity NH₄⁺ uptake. Figure 4 shows how NH₄⁺ uptake by rice plants is affected by prior growth on 2 m m (G2), 100 m m (G100) or 1000 m m (G1000) NH₄⁺ before measuring ¹³NH₄⁺ influx.

Figure 4

Using Arabidopsis plants the same response was obtained for plants grown on 0.1 mM, 1 mM, or 10 mM NH₄⁺ prior to measuring ¹³NH₄⁺ influx. On Fig. 5 you can see this result and above the influx plot you can see a Northern blot of AMT1 expression in the same root tissue.

Figure 5

When Arabidopsis roots that were starved of NH₄⁺ for 2 days were re-supplied with NH₄⁺ (Fig. 6) both ¹³NH₄⁺ influx and AMT1 gene expression declined.

Figure 6

The question we set out to answer was "Is it NH₄⁺ which regulates AMT1 expression, or is it a product of ammonium assimilation?" To answer this question we repeated the experiment using the metabolic inhibitor MSX which blocks the conversion of NH₄⁺ to the amino acid glutamine. When MSX was applied Arabidopsis roots that had been starved of NH₄⁺ for 2 days then re-supplied with NH₄⁺, the MSX - treated roots had 30 times higher NH₄⁺ but much lower levels of glutamine. These roots showed virtually no decrease of NH₄⁺ influx or AMT1 expression (Fig. 7).

Figure 7

Progress has also been made with the high affinity nitrate transporter. In Aspergillus a mutant defective in NO₃^- was isolated some time ago. The gene responsible for this defect was named crnA. Last year a group in Spain isolated mutants from Chlamydomonas which were defective in nitrate uptake. Three gene loci (nar-2, nar-3, and nar-4) are present as a gene cluster, including nit-1 (the NR structural gene), all regulated by nitrate were identified. It was deduced that nar-2 plus nar-3 , or nar-2 plus nar-4 could support nitrate transport but not single genes. The nar genes are homologous to the Asp crnA gene. At Rothamsted, a British group used the Aspergillus sequence to fish out a homologous sequence from barley. We have isolated these genes from Arabidopsis and other similar genes from barley. In barley they have been named pBCH1, pBCH2, and pBCH3. We are presently trying to understand why as many as 10 genes encode pBCH homologs in barley and Arabidopsis.

3.Response to inadequacy : cytoplasm or vacuole ?

An important question that remains unclear is the signal to increase expression of the transporter genes. The cytoplasm is a very small compartment and should respond very rapidly to withholding a particular ion. Hence it could be hypothesized that withholding a particular ion reduces cytoplasmic concentration of that ion and this change is somehow transmitted to the gene level to cause increased transcription. However, several studies indicate that withholding an ion simply causes the vacuolar concentration to decline while the cytosolic concentration remains constant. In Fig 8 the data of cytoplasmic Pi is plotted against vacuolar Pi during several days of growth in solutions containing zero Pi. It is evident that cytoplasmic Pi remains constant at the expense of vacuolar Pi. Likewise, we have used compartmental analysis to estimate cytoplasmic [K⁺] in barley plants grown at different levels of K⁺. The message is the same : cytoplasmic ion concentration remains constant while vacuole reserves are used up. It would appear that the vacuole is the site of change and perhaps, therefore, it is the source of the signal evoking the transcriptional response.

Figure 8

4. Cycling of nutrients in the phloem.

A current model for the regulation of root ion uptake activity in response to whole plant demand, is that nutrients cycle between the leaves and root via the phloem. Ions are already transferred to the xylem from the root, so this is a two-way transport: up in the xylem and back in the phloem. If the plant has a plentiful supply of a given element, the concentration of that ion in the return flow in the phloem will be high. This signals (we don't know how exactly) that the demand for uptake is low. On the other hand if the plant is short of an element then its concentration in the return flow will be low and this signifies to the root that uptake should be increased. Read the review by Marschner et al.

Other problems.

When plants are deprived of a particular nutrient, a typical response is for the root: shoot ratio to increase. We have observed consistently that the transport of that ion to the shoot is greatly reduced under these conditions, and does not begin again until about 6 h after the supply of nutrients is restored. This is another important area for study about which we know very little.

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